Methods of using compositions comprising variants and fusions of FGF19 polypeptides for treatment of nonalcoholic steatohepatitis

ABSTRACT

The invention relates to variants and fusions of fibroblast growth factor 19 (FGF19), variants and fusions of fibroblast growth factor 21 (FGF21), fusions of FGF19 and/or FGF21, and variants or fusions of FGF19 and/or FG-F21 proteins and peptide sequences (and peptidomimetics), having one or more activities, such as bile acid homeostasis modulating activity, and methods for and uses in treatment of bile acid and other disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371 national stage application of internationalapplication Serial No. PCT/US2013/077782 filed Dec. 26, 2013, whichclaims the benefit of U.S. Ser. No. 61/746,499 filed Dec. 27, 2012, U.S.Ser. No. 61/779,604 filed Mar. 13, 2013, and U.S. Ser. No. 61/887,129filed Oct. 4, 2013, each of which is incorporated herein by reference inits entirety.

FIELD OF THE INVENTION

The invention relates to variants of fibroblast growth factor 19 (FGF19)proteins and peptide sequences (and peptidomimetics) and fusions ofFGF19 and/or fibroblast growth factor 21 (FGF21) proteins and peptidesequences (and peptidomimetics), and variants of fusions of FGF19 and/orFGF21 proteins and peptide sequences (and peptidomimetics) that modulatebile acid homeostasis, and methods for and uses of the variants andfusions in treatment of bile acid related and associated disorders.

INTRODUCTION

Bile acids, steroid acids that are found predominantly in the bile ofmammals, regulate cholesterol, triglyceride, glucose and energyhomeostasis, and facilitate digestion and absorption of lipids in thesmall intestine. Emulsification of lipids and fat-soluble vitamins inthe intestine allows the formation of micelles that can then betransported via the lacteal system. Other functions of bile acidsinclude driving the flow of bile to eliminate catabolites from the liverand aiding in the reduction of the bacteria flora found in the smallintestine and biliary tract. Bile acids are also involved in theregulation of their own synthesis and enterohepatic circulation. See,e.g., Staels et al., Diabetes Care (2009) vol. 32 no. suppl 2 S237-S245.

In humans, bile acid production occurs primarily in the perivenoushepatocytes through a series of enzymatic reactions that convertcholesterol into the two primary bile acids, cholic acid andchenodeoxycholic acid. The primary bile acids are synthesized by twodistinct pathways. In the “classic” or “neutral” pathway, the primarybile acids are produced by hydroxylation of cholesterol throughcatalysis by the cytochrome P450 enzyme cholesterol 7α-hydroxylase(cyp7a1), which catalyzes the first and rate-limiting step in theclassical bile acid synthesis pathway. (See, e.g., Inagaki et al., CellMetabolism 2:217-25 (October 2005)).

As described further herein, activity of cyp7a1 is down-regulated bycholic acid and up-regulated by cholesterol; thus, cyp7a1 is regulatedby bile acids themselves. The conversion of cholesterol to bile acids isprimarily effected by this pathway. In addition, in most individualsapproximately 6% of bile acids are synthesized by an “alternative” or“acidic” pathway. This pathway is regulated by the enzyme cyp27a1, whichconverts oxysterols to bile acids. In contrast to cyp7a1, cyp27a1 is notregulated by bile acids themselves.

When cholic acid and chenodeoxycholic acid are secreted into the lumenof the intestine, intestinal bacteria dehydroxylate a portion of each toform the secondary bile acids, deoxycholic acid (derived from cholicacid) and lithocholic acid (derived from chenodeoxycholic acid). Hepaticcells may conjugate these four bile with one of two amino acids, glycineor taurine, to form a total of eight possible conjugated bile acids,referred to as bile salts. Thus, in total the principal bile acids arecholic acid, chenodeoxycholic acid, glycocholic acid, taurocholic acid,deoxycholic acid and lithocholic acid. All four of these bile acids canbe transported back into the blood stream, be returned to the liver, andbe re-secreted through enterohepatic circulation. See, e.g., Staels etal., Diabetes Care (2009) vol. 32 suppl 2 S237-S245.

The primary bile acids (cholic acid and chenodeoxycholic acid) aresynthesized in the liver), while the secondary bile acids (deoxycholicacid and lithocholic acid) are made by bacteria. The four bile acids aresecreted into the bile canalicular lumen for storage in the gallbladderas mixed micelles with phospholipids and cholesterol. Upon ingestion ofa meal, cholecystokinin stimulates gallbladder contraction resulting inits release of micellar bile acids into the intestinal lumen to aiddigestion. Enterohepatic circulation enables ˜90-95% of bile acids to bereabsorbed from the distal ileum and transported back to the liver; thisbile acid uptake and transportation occurs primarily by pericentralhepatocytes. The approximately 5% of bile acids that are not reabsorbedare eliminated in the feces, and that amount of loss is subsequentlyreplaced by de novo bile acid synthesis in the liver. See, e.g., Rose etal., Cell Metabolism, 14:1, pp 123-130 (6 Jul. 2011).

The primary bile acids (chenodeoxycholic acid and cholic acid) arephysiological ligands/activators of farnesoid-X-receptor (FXR),pregnane-X-receptor (PXR) and constitutive androstane receptor (CAR),and litocholic acid is a ligand for the Vitamin D receptor (VDR) and theG-protein coupled receptor TGR5. FXR demonstrates a high selectivity forbile acids; conversely, PXR and CAR act upon a number of receptorsintegrating lipid homeostasis with xenobiotic metabolism. FXR, PXR, CARand TGR5 exert synergistic activities in regulating lipid and glucosehomeostasis and energy expenditure, as well as in regulating liver andperipheral insulin sensitivity. As surfactants or detergents, bile acidsare potentially toxic to cells, and the size of the bile acid pool istightly regulated within the liver and intestine to prevent cytotoxicaccumulation. When the bile acid pool size increases, a feedbackmechanism involving the interplay of several nuclear receptors,including FXR, is activated to inhibit de novo bile acid synthesis. See,e.g., Fiorucci et al., Prog Lipid Res. 2010 April; 49(2):171-85. Epub2009 Dec. 2.

The synthesis of bile acids in the liver is negatively regulated by thehormone FGF19. FGF19 is secreted from the intestine and signals to theliver to repress Cyp7a1. In comparison, intestinal FXR activation due totransintestinal bile acid flux after a meal also induces the expressionof FGF19, which is released by small intestine epithelial cells andcirculates to bind to hepatocyte FGF receptor 4 (FGFR4) receptors; theFGFR4 receptors signal a reduction in bile acid synthesis via c-JunNH₂-terminal kinase (INK) pathway activation. Repression of CYP7A1results in decreased synthesis of bile acids from intrahepaticcholesterol in response to the daily feeding-fasting cycle.

Therapeutic Implications

As described herein, abnormal bile acid homeostasis can result in, orexacerbate, a number of disorders, including cholestasis, portosystemicshunt, Crohn's disease, and hepatic microvascular dysplasia. Inaddition, bile acids play a role in modulating the metabolic syndrome, acluster of cardiovascular disease risk factors that include visceralobesity, insulin resistance, dyslipidemia, increased blood pressure, andhypercoagulability. Thus, modulation of bile acid activity can provide anumber of beneficial therapeutic effects.

Lipid- and Glucose-Related Disorders

Activation of FXR by bile acids (or nonsteroidal synthetic FXR agonists)lowers plasma triglycerides and has been shown to improve hyperglycemiain diabetic mice. Bile acids may also regulate energy expenditure in anFXR-independent manner in mice through activation of the Gprotein-coupled receptor TGR5. Thus, modulation of FXR activity and bileacid metabolism may provide a therapeutic approach for the treatment of,for example, the metabolic syndrome and diabetes type 2. See, e.g.,Lefebvre et al., Physiol Rev. 2009 January; 89(1):147-91.

Bile acid synthesis (along with ileal resection) disrupts theenterohepatic circulation of bile acids, decreases plasma total and LDLcholesterol, and increases levels of HDL cholesterol, apolipoprotein(apo)-AI, and triglycerides. As a direct consequence of interrupting thereturn of bile acids to the liver, cyp7a1 expression becomesde-repressed, and conversion of cholesterol into bile acids isstimulated. Thus, agents that sequester bile acids in the gut (e.g.,cholestyramine) prevent their reabsorption, resulting in, as acompensatory mechanism, more endogenous cholesterol being shunted intothe production of bile acids, leading to reduced cholesterol levels.

The depletion of hepatic cholesterol due to increased diversion to bileacid synthesis leads to increased hepatic LDL receptor expression, whichresults in LDL receptor expression that accounts for the decline intotal and LDL cholesterol produced by bile acid synthesis or ilealresection. There is thought to be an independent regulatory role for FXRin both HDL cholesterol and triglyceride metabolism.

As noted, bile acid synthesis has also been found to be associated withtype 2 diabetes. A number of factors may contribute to glucoseregulation, including effects on bile acid pool size and composition,FXR-mediated alterations in hepatic glucose production and intestinalglucose absorption, influences on peripheral insulin sensitivity,incretin effects, and energy use. Not only is modulation of bile acidsynthesis useful in the treatment of diabetes, it may also find clinicalutility in the treatment of pre-diabetes.

Bile Acid Malabsorption and Diarrhea

Excess concentrations of bile acids in the colon, resulting from, forexample, bile acid malabsorption, are a cause of chronic diarrhea. Whenlarge amounts of bile acids enter the colon, they stimulate watersecretion and intestinal motility causing chronic diarrhea, a conditionreferred to as a bile acid diarrhea (BAD). More particularly, whenintestinal expression of the bile acid transporters is reduced, theintestine is less efficient at bile acid reabsorption (Type 1 bile acidmalabsorption). Similarly, if intestinal motility is affected bygastro-intestinal surgery, or bile acids are deconjugated by smallintestinal bacterial overgrowth, absorption is less efficient (Type 3bile acid malabsorption). There is also a very small group of patientswhich do not exhibit any obvious signs of disease (Type 2 bile acidmalabsorption). (See generally, Walters et al., Clin. GastroenterolHepatol. 7:1189-94 (November 2009)).

Cholestasis and Primary Biliary Cirrhosis

The condition of cholestasis is caused by acute or chronic interruptionin the excretion of bile (through, for example, obstruction) within oroutside the liver. Failure to form bile results in progressivecholestatic liver injury and death. Obstruction causes bile salts, thebile pigment bilirubin, and lipids to accumulate in the blood streaminstead of being eliminated normally. Symptoms of chronic cholestasisinclude skin discoloration, scars or skin injuries caused by scratching,bone pain, xanthoma, or xanthelasma. Patients with advanced cholestasisfeel ill, tire easily, and are often nauseated. Abdominal pain and suchsystemic symptoms as anorexia, vomiting, and fever are usually due tothe underlying condition that causes cholestasis.

Intrahepatic cholestasis is usually caused by hepatitis or bymedications that produce symptoms resembling hepatitis.Phenothiazine-derivative agents, including chlorpromazine, can causesudden fever and inflammation, although symptoms usually disappear aftercessation of the agents. In rare cases, a condition resembling chronicbiliary cirrhosis, discussed further below, persists even after themedication is stopped. Some patients experience a similar reaction inresponse to, for example, tricyclic antidepressants (e.g., amitriptylineand imipramine) and phenylbutazone. Intrahepatic cholestasis may alsohave other causes, including alcoholic liver disease, primary biliarycirrhosis, and cancer that has metastasized.

In comparison, there are several origins of extrahepatic cholestasis,including as an adverse effect of certain medications, a complication ofsurgery, serious injury, tissue-destroying infection, or intravenousfeeding. Extrahepatic cholestasis can be caused by conditions such astumors and gallstones that block the flow of bile from the gallbladderto the duodenum (e.g., by a stone obstructing the common bile duct).Extrahepatic cholestasis may also be caused by pancreatic cancer and,less frequently, as a result of non-cancerous narrowing of the commonduct, ductal carcinoma, or disorders of the pancreas.

Symptoms of both intrahepatic and extrahepatic cholestasis includejaundice, dark urine, and pale stools. Itching over the skin may besevere if the condition is advanced.

Intrahepatic cholestasis of pregnancy (ICP) frequently develops duringthe second and third trimesters of pregnancy, and it is the second mostcommon cause of jaundice during pregnancy. Although symptoms usuallydisappear within two-to-four weeks after the baby's birth, they mayreappear if the mother subsequently becomes again. A similar conditionaffects some women who take oral contraceptives, but symptoms disappearupon cessation of the use of oral contraceptives.

Inborn errors of bile acid synthesis are rare genetic disorders thatsometimes present as neonatal cholestasis. It is characterized by afailure to produce normal bile acids and an accumulation of unusual bileacids and bile acid intermediates. If not diagnosed or if diagnosedimproperly, such inborn errors can result in liver failure orprogressive chronic liver disease.

Drug-induced cholestasis may be a complication of chemotherapy or othermedications. The two major types of drug-induced cholestasis areidiosyncratic reactions and direct toxic injury. Idiosyncratic reactionsmay occur at the onset of treatment or thereafter. Allergic responsesare varied and are not related to the amount of medication being taken.

In direct toxic injury, the severity of symptoms parallels the amount ofmedication involved. This condition develops a short time aftertreatment begins, follows a predictable pattern, and usually causesliver damage. Direct toxic reactions develop in 1% of all patients whotake chlorpromazine.

The rare condition of benign familial recurrent cholestasis ischaracterized by brief, repeated episodes of itching and jaundice,although the symptoms frequently disappear and the condition does notcause cirrhosis. (See generally, Rose et al., Cell Metabolism14(1):123-30 (July 2011).

Primary Biliary Cirrhosis (PBC) is a progressive hepatic disease thatprimarily results from autoimmune destruction of the bile ducts thattransport bile acids out of the liver, resulting in cholestasis. As thedisease progresses, persistent toxic build-up of bile acids causesprogressive liver damage marked by chronic inflammation and fibrosis.

While PBC is rare, it is the most common cholestatic liver disease andis the fifth most common cause of liver transplant in the United States.A majority of PBC patients are asymptomatic at the time of initialdiagnosis, but most develop symptoms, such as fatigue and pruritus, overtime. Jaundice may result from advanced disease. Though not required, aliver biopsy can be used to confirm the diagnosis of PBC, and bilirubinis frequently monitored to provide an indication of liver function.Elevated serum levels of ALP, an enzyme released by hepatic cells inresponse to bile acid-mediated toxicity, is generally closely monitoredin patients as an indicator of treatment response and prognosis.

Despite receiving ursodiol, the standard of care therapy for PBC, asignificant portion of patients at advanced stated PBC will progress toliver failure, transplant or death within five-ten years. As a result,alternative therapies are currently being evaluated. One potentiallypromising agent is OCA, is a bile acid analog and FXR agonist derivedfrom the primary human bile acid chenodeoxycholic acid, or CDCA. OCA isbeing evaluated for patients having an inadequate therapeutic responseto ursodiol or who are unable to tolerate ursodiol (InterceptPharmaceuticals, New York).

Primary Sclerosing Cholangitis

Primary sclerosing cholangitis is a chronic fibrosing inflammatoryprocess that results in the destruction of the biliary tree and biliarycirrhosis. The strictures are located in both the intrahepatic andextrahepatic ducts in more than 80% of the patients, but about 10% ofthese patients have only intrahepatic strictures, while less than 5%will have only extrahepatic strictures. Remissions and relapsescharacterize the disease course. Although the cause of primarysclerosing cholangitis is unknown, it is believed that damage to thebile duct occurs through one or more of genetic abnormalities of immuneregulation, viral infection, toxins from intestinal bacteria, bacteriain the portal venous system, ischemic vascular damage, and toxic bileacids from intestinal bacteria.

The majority of patients with primary sclerosing cholangitis haveunderlying inflammatory bowel disease (ulcerative colitis or Crohn'sdisease). Patients are more likely to have ulcerative colitis thanCrohn's disease (85% versus 15%), with approximately 2.5-7.5% of allulcerative colitis patients having primary sclerosing cholangitis.Primary sclerosing cholangitis may remain quiescent for long periods oftime in some patients; in most cases, however, it is progressive.

The prevalence of primary sclerosing cholangitis in the United States isapproximately 1-6 cases per 100,000 population, and the vast majorityare Caucasian. Approximately 75% of patients with primary sclerosingcholangitis are men having an average age of approximately 40 years atthe time of diagnosis. Management of this disease in the early stagesinvolves the use of drugs to prevent disease progression. Endoscopic andsurgical approaches are reserved for the time when symptoms develop.Liver transplantation may ultimately be required and offers the onlychance for a complete cure. Patients with primary sclerosing cholangitisare at an increased risk for cholangiocarcinoma (10-15%).

Most patients with primary sclerosing cholangitis do not exhibitsymptoms and are usually diagnosed by the detection of abnormalbiochemical tests of liver function on routine blood testing. Whensymptoms develop they are a result of obstruction to bile flow andinclude jaundice, itching, right upper quadrant abdominal pain, fever,and chills. Symptoms may also include weight loss and fatigue. Patientsmay remain asymptomatic for many years despite the presence of advanceddisease, and the development of symptoms usually suggests the presenceof advanced disease.

Diagnosis

Bile acid malabsorption is readily diagnosed by the SeHCAT(23-seleno-25-homo-tauro-cholic acid (selenium homocholic acid taurineor tauroselcholic acid)) nuclear medicine test. An alternativediagnostic test involves measurement in the serum of 7alpha-hydroxy-4-cholesten-3-one, a bile acid precursor.

Treatment

Bile acid sequestrants (e.g., cholestyramine and colestipol which are inpowder form) are the main agents used to treat bile acid malabsorption.Unfortunately, many patients do not tolerate cholestyramine andcolestipol, often because of the poor texture and taste of the resinpowder. Fortunately, the bile acid sequestrant colesevelam is availablein tablet form and is often better tolerated.

All bile acid sequestrants are capable of binding other compounds, andit is also possible that deficiencies of fat-soluble vitamins (A, D, Eand K) may occur, requiring administration of vitamin supplements.

Displacement and replacement therapy have also proven useful in certaindisorders associated with bile acid homeostasis. In displacementtherapy, the composition of the circulating bile acids is changed,either to decrease the cytotoxicity of endogenous bile acids or tomodulate cholesterol metabolism to decrease biliary cholesterolsecretion. Conversely, bile acid replacement aims to correct a bile aciddeficiency.

Displacement Therapy

Administration of the primary bile acid chenodeoxycholic Acid (CDCA) hasbeen shown to decrease in biliary cholesterol secretion and gradualdissolution of gallstones. CDCA was gradually replaced byursodeoxycholic acid (UDCA) because the later did not result in anyhepatotoxicity. Chenodeoxycholic acid is slightly hepatotoxic in humans,but in certain animals, it is highly hepatotoxic. Despite the efficacyand safety of UDCA administration for cholesterol gallstone dissolution,it is not frequently used today because of the success of laparoscopiccholecystectomy, which provides a rapid cure for symptomatic disease.Medical therapy, in contrast, requires months of therapy, does notalways dissolve stones, and is followed by gradual recurrence in somepatients.

UDCA therapy has been shown to improve liver test results in patientswith primary biliary cirrhosis, an effect that likely involves multiplemechanisms. UDCA therapy has also shown favorable effects in othercholestatic conditions, such as cholestasis associated with pregnancyand cholestasis associated with total parenteral nutrition.

Replacement Therapy

Bile acid replacement is used in inborn errors of bile acidbiosynthesis, usually with a mixture of chenodeoxycholic Acid (CDCA) orUrsodeoxycholic Acid (UDCA) and cholic acid, to suppress the synthesisof cytotoxic bile acid precursors and restore the input of primary bileacids into the enterohepatic circulation.

In patients with a short-bowel syndrome, a bile acid deficiency occursin the proximal intestine, leading to impaired micellar solubilization.This, plus the decreased surface area and rapid transit time, leads tosevere fat malabsorption. Cholylsarcosine (cholyl-N-methylglycine), asynthetic bile acid analogue, has been shown to increase lipidabsorption in a patient with short-bowel syndrome, and it is resistantto deconjugation and dehydroxylation.

Patients with bile acid diarrhea secondary to Crohn's ileitis will behelped with glucocorticoid treatment, and microscopic colitis is alsohelped by steroids. Administration of budesonide and other agents,including antibiotics, are useful in certain situations.

As detailed above, treatment of PBC generally entails administration ofursodiol, though alternative therapies are being evaluated for patientshaving an inadequate therapeutic response to ursodiol or who are unableto tolerate ursodiol.

Accordingly, there is a need for treatment of bile acid disorders, suchas the foregoing disorders and including, but not limited to: metabolicsyndrome; a lipid or glucose disorder; cholesterol or triglyceridemetabolism; type 2 diabetes; cholestasis, including, for examplediseases of intrahepatic cholestasis (e.g., primary biliary cirrhosis(PBC), primary familial intrahepatic cholestasis (PFIC) (e.g.,progressive PFIC), primary sclerosing choangitis (PSC), pregnancyintrahepatic cholestasis (PIC), neonatal cholestasis, and drug inducedcholestasis (e.g., estrogen)), and diseases of extrahepatic cholestasis(e.g., bile cut compression from tumor, bile duct blockade by gallstones); bile acid malabsorption and other disorders involving thedistal small intestine, including ileal resection, inflammatory boweldiseases (e.g., Crohn's disease and ulcerative colitis), disordersimpairing absorption of bile acids not otherwise characterized(idiopathic)) leading to diarrhea (e.g., bile acid diarrhea (BAD)) andGI symptoms, and GI, liver, and/or biliary cancers (e.g., colon cancerand hepatocellular cancer); and/or bile acid synthesis abnormalities,such as those contributing to non-alcoholic steatohepatitis (NASH),cirrhosis and portal hypertension; e.g., in mammals, such as humans. Theinvention satisfies this need and provides related benefits.

SUMMARY

The invention is based, in part, on variants of FGF19 peptide sequences,fusions of FGF19 and/or FGF21 peptide sequences and variants of fusions(chimeras) of FGF19 and/or FGF21 peptide sequences having one or moreactivities, such as bile acid homeostasis modulating activity. Suchvariants and fusions (chimeras) of FGF19 and/or FGF21 peptide sequencesinclude sequences that are used for treating a bile-acid related orassociated disorder. Such variants and fusions (chimeras) of FGF19and/or FGF21 peptide sequences also include sequences that do notsubstantially or significantly increase or induce hepatocellularcarcinoma (HCC) formation or HCC tumorigenesis. Such variants andfusions (chimeras) of FGF19 and/or FGF21 peptide sequences furtherinclude sequences that do not induce a substantial elevation or increasein lipid profile.

In one embodiment, a method or use of modulating bile acid homeostasisor treating a bile-acid related or associated disorder includes:administering a chimeric peptide sequence, comprising: a) an N-terminalregion comprising at least seven amino acid residues, the N-terminalregion having a first amino acid position and a last amino acidposition, wherein the N-terminal region comprises DSSPL or DASPH; and b)a C-terminal region comprising a portion of SEQ ID NO:99 [FGF19], theC-terminal region having a first amino acid position and a last aminoacid position, wherein the C-terminal region comprises amino acidresidues 16-29 of SEQ ID NO:99 [FGF19] (WGDPIRLRHLYTSG; SEQ ID NO:169),wherein the W residue corresponds to the first amino acid position ofthe C-terminal region, to modulate bile acid homeostasis or treat thebile-acid related or associated disorder.

In another embodiment, a method or use of modulating bile acidhomeostasis or treating a bile-acid related or associated disorderincludes: administering a chimeric peptide sequence, comprising: a) anN-terminal region comprising a portion of SEQ ID NO:100 [FGF21], theN-terminal region having a first amino acid position and a last aminoacid position, wherein the N-terminal region comprises amino acidresidues GQV, and wherein the V residue corresponds to the last aminoacid position of the N-terminal region; and b) a C-terminal regioncomprising a portion of SEQ ID NO:99 [FGF19], the C-terminal regionhaving a first amino acid position and a last amino acid position,wherein the C-terminal region comprises amino acid residues 21-29 of SEQID NO:99 [FGF19], RLRHLYTSG (SEQ ID NO:185), and wherein the R residuecorresponds to the first position of the C-terminal region, to modulatebile acid homeostasis or treat the bile-acid related or associateddisorder.

In a further embodiment, a method or use of modulating bile acidhomeostasis or treating a bile-acid related or associated disorderincludes: administering a chimeric peptide sequence, comprising: a) anN-terminal region comprising a portion of SEQ ID NO:100 [FGF21], theN-terminal region having a first amino acid position and a last aminoacid position, wherein the N-terminal region comprises at least 5contiguous amino acids of SEQ ID NO:100 [FGF21] including the amino acidresidues GQV, and wherein the V residue corresponds to the last aminoacid position of the N-terminal region; and b) a C-terminal regioncomprising a portion of SEQ ID NO:99 [FGF19], the C-terminal regionhaving a first amino acid position and a last amino acid position,wherein the C-terminal region comprises amino acid residues 21-29 of SEQID NO:99 [FGF19], RLRHLYTSG (SEQ ID NO:185), and wherein the R residuecorresponds to the first position of the C-terminal region, to modulatebile acid homeostasis or treat the bile-acid related or associateddisorder.

In an additional embodiment, a method or use of modulating bile acidhomeostasis or treating a bile-acid related or associated disorderincludes: administering a peptide sequence, comprising or consisting ofany of: a) a FGF19 sequence variant having one or more amino acidsubstitutions, insertions or deletions compared to a reference or wildtype FGF19; b) a FGF21 sequence variant having one or more amino acidsubstitutions, insertions or deletions compared to a reference or wildtype FGF21; c) a portion of an FGF19 sequence fused to a portion of anFGF21 sequence; or d) a portion of an FGF19 sequence fused to a portionof an FGF21 sequence, wherein the FGF19 and/or FGF21 sequence portion(s)have one or more amino acid substitutions, insertions or deletionscompared to a reference or wild type FGF19 and/or FGF21, to modulatebile acid homeostasis or treat the bile-acid related or associateddisorder.

In various particular embodiments, a chimeric peptide sequence has anN-terminal region with at least 6 contiguous amino acids of SEQ IDNO:100 [FGF21] including the amino acid residues GQ; or has anN-terminal region with at least 7 contiguous amino acids of SEQ IDNO:100 [FGF21] including the amino acid residues GQV.

In various additional embodiments, a peptide sequence has amino-terminalamino acids 1-16 of SEQ ID NO:100 [FGF21] fused to carboxy-terminalamino acids 21-194 of SEQ ID NO:99 [FGF19], or the peptide sequence hasamino-terminal amino acids 1-147 of SEQ ID NO:99 [FGF19] fused tocarboxy-terminal amino acids 147-181 of SEQ ID NO:100 [FGF21] (M41), orthe peptide sequence has amino-terminal amino acids 1-20 of SEQ ID NO:99[FGF19] fused to carboxy-terminal amino acids 17-181 of SEQ ID NO:100[FGF21] (M44), or the peptide sequence has amino-terminal amino acids1-146 of SEQ ID NO:100 [FGF21] fused to carboxy-terminal amino acids148-194 of SEQ ID NO:99 [FGF19] (M45), or the peptide sequence hasamino-terminal amino acids 1-20 of SEQ ID NO:99 [FGF19] fused tointernal amino acids 17-146 of SEQ ID NO:100 [FGF21] or fused tocarboxy-terminal amino acids 148-194 of SEQ ID NO:99 [FGF19] (M46).

In various further embodiments, a peptide sequence has at least oneamino acid substitution to amino acid residues 125-129 of SEQ ID NO:99[FGF19], EIRPD; at least one amino acid substitution to amino acidresidues 126-128 of SEQ ID NO:99 [FGF19], IRP; or at least one aminoacid substitution to amino acid residues 127-128 of SEQ ID NO:99[FGF19], RP, or at least one amino acid substitution to amino acidresidues 1-124 of SEQ ID NO:99 [FGF19] and/or to amino acid residues130-194 of SEQ ID NO:99 [FGF19]. More specifically, for example, apeptide sequence with a substitution to one of amino acid residues127-128 of SEQ ID NO:99 [FGF19], IRP, wherein at least one amino acidsubstitution is R127L or P128E.

Methods and uses of the invention can be practiced using a peptide orchimeric sequence, as set forth herein. For example, a sequence thatincludes or consists of any peptide sequence set forth herein as M1 toM98, or M101 to M160, or SEQ ID NOs:1 to 98, 101 to 135, or 138 to 196,a peptide sequence that includes or consists of any sequence set forthin Tables 1-10, or a peptide sequence that includes or consists of anysequence set forth in the Sequence Listing herein.

Methods and uses of the invention can be practiced using a peptide orchimeric sequence of any suitable length. In particular embodiments, theN-terminal or C-terminal region of the peptide or chimeric sequence isfrom about 20 to about 200 amino acid residues in length. In otherparticular aspects, a peptide or chimeric sequence has 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino aciddeletions from the amino terminus, the carboxy-terminus or internally.In further particular embodiments, a peptide or chimeric sequence has anN-terminal region, or a C-terminal region that includes or consists ofan amino acid sequence of about 5 to 10, 10 to 20, 20 to 30, 30 to 40,40 to 50, 60 to 70, 70 to 80, 80 to 90, 90 to 100 or more amino acids.In additional more particular embodiments, a peptide or chimericsequence has an FGF19 sequence portion, or an FGF21 sequence portionthat includes or consists of an amino acid sequence of about 5 to 10, 10to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to90, 90 to 100 or more amino acids of FGF19 or FGF21.

In various aspects, a peptide sequence has: a WGDPI (SEQ ID NO:170)sequence motif corresponding to the WGDPI sequence of amino acids 16-20of SEQ ID NO:99 [FGF19]; has a substituted, mutated or absent WGDPI (SEQID NO:170) sequence motif corresponding to FGF19 WGDPI (SEQ ID NO:170)sequence of amino acids 16-20 of FGF19; has a WGDPI (SEQ ID NO:170)sequence with one or more amino acids substituted, mutated or absent. Invarious other further aspects, the peptide sequence is distinct from anFGF 19 variant sequence having any of GQV, GDI, WGPI (SEQ ID NO:171),WGDPV (SEQ ID NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ ID NO:174), GPI,WGQPI (SEQ ID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQ ID NO:177),WADPI (SEQ ID NO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ ID NO:180),WDPI (SEQ ID NO:181), WGDI (SEQ ID NO:182), WGDP (SEQ ID NO:183) orFGDPI (SEQ ID NO:184) substituted for the FGF19 WGDPI (SEQ ID NO:170)sequence at amino acids 16-20.

In various further aspects, the N-terminal region comprises amino acidresidues VHYG (SEQ ID NO:101), wherein the N-terminal region comprisesamino acid residues DASPHVHYG (SEQ ID NO:102), or the N-terminal regioncomprises amino acid residues DSSPLVHYG (SEQ ID NO:103). Moreparticularly, in one aspect the G corresponds to the last position ofthe N-terminal region.

In various additional aspects, the N-terminal region comprises aminoacid residues DSSPLLQ (SEQ ID NO:104), where the Q residue is the lastamino acid position of the N-terminal region, or comprises amino acidresidues DSSPLLQFGGQV (SEQ ID NO:105), where the V residue correspondsto the last position of the N-terminal region.

More particularly, an N-terminal region further includes: RHPIP (SEQ IDNO:106), where R is the first amino acid position of the N-terminalregion; or HPIP (SEQ ID NO:107), where H is the first amino acidposition of the N-terminal region; or RPLAF (SEQ ID NO:108), where R isthe first amino acid position of the N-terminal region; or PLAF (SEQ IDNO:109), where P is the first amino acid position of the N-terminalregion; or R, where R is the first amino acid position of the N-terminalregion.

In various other aspects, a peptide or chimeric sequence has: amino acidresidues HPIP (SEQ ID NO:107), which are the first 4 amino acid residuesof the N-terminal region. In various still further aspects, a peptide orchimeric sequence has: an R residue at the first position of theN-terminal region, or the first position of the N-terminal region is anM residue, or the first and second positions of the N-terminal region isan MR sequence, or the first and second positions of the N-terminalregion is an RM sequence, or the first and second positions of theN-terminal region is an RD sequence, or the first and second positionsof the N-terminal region is an DS sequence, or the first and secondpositions of the N-terminal region is an MD sequence, or the first andsecond positions of the N-terminal region is an MS sequence, or thefirst through third positions of the N-terminal region is an MDSsequence, or the first through third positions of the N-terminal regionis an RDS sequence, or the first through third positions of theN-terminal region is an MSD sequence, or the first through thirdpositions of the N-terminal region is an MSS sequence, or the firstthrough third positions of the N-terminal region is an DSS sequence, orthe first through fourth positions of the N-terminal region is an RDSS(SEQ ID NO:115), sequence, or the first through fourth positions of theN-terminal region is an MDSS (SEQ ID NO:116), sequence, or the firstthrough fifth positions of the N-terminal region is an MRDSS (SEQ IDNO:117), sequence, or the first through fifth positions of theN-terminal region is an MSSPL (SEQ ID NO:113) sequence, or the firstthrough sixth positions of the N-terminal region is an MDSSPL (SEQ IDNO:110) sequence, or the first through seventh positions of theN-terminal region is an MSDSSPL (SEQ ID NO:111) sequence.

In various other particular aspects, a peptide or chimeric sequence hasat the N-terminal region first amino acid position an “M” residue, an“R” residue, a “S” residue, a “H” residue, a “P” residue, a “L” residueor an “D” residue. In various alternative particular aspects, a peptideor chimeric sequence peptide sequence does not have a “M” residue or an“R” residue at the first amino acid position of the N-terminal region.

In further various other aspects, a peptide or chimeric sequence has anN-terminal region with any one of the following sequences: MDSSPL (SEQID NO:110), MSDSSPL (SEQ ID NO:111), SDSSPL (SEQ ID NO:112), MSSPL (SEQID NO:113) or SSPL (SEQ ID NO:114).

In various still additional aspects, a peptide or chimeric sequence hasa residue at the last position of the C-terminal region that correspondsto about residue 194 of SEQ ID NO:99 [FGF19].

In various more particular aspects, a peptide sequence has or consistsof any one of the following sequences:

(SEQ ID NO: 3)RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M3); (SEQ ID NO: 194)RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIREDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M140); (SEQ ID NO: 196)RPLAFSDAGPHVHYGWGDPIRQRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M160); (SEQ ID NO: 69)RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M69); (SEQ ID NO: 52)RDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K (M52);(SEQ ID NO: 5)RHPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M5); (SEQ ID NO: 160)HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M5-R); (SEQ ID NO: 71)HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHSLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS (M71);(SEQ ID NO: 72)HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS (M72);(SEQ ID NO: 73)HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVVQDELQGVGGEGCHMHPENCKTLLTDIDRTHTEKPVWDGITGE (M73); (SEQ ID NO: 1 or 139)RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M1); (SEQ ID NO: 2 or 140)RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M2); (SEQ ID NO: 48 or 6 or 148)RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K (M48);(SEQ ID NO: 49 or 7 or 149)RPLAFSDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M49); (SEQ ID NO: 50)RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M50); (SEQ ID NO: 51 or 36 or 155)RHPIPDSSPLLQFGGNVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M51); (SEQ ID NO: 192)MDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K (M53);(SEQ ID NO: 70)MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDS16MDPFGLVTGLEAVRSPSFEK (M70); (SEQ ID NO: 193)RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M139); or (SEQ ID NO: 195)RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILCDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M141);or a subsequence or fragment thereof of any of the foregoing peptidesequences. In certain embodiments of any of the foregoing peptidesequences, the R terminal residue is deleted.

In various additional particular aspects, the N-terminus of the peptidesequence includes or consists of any of:

HPIPDSSPLLQFGGQVRLRHLYTSG (M5-R) (amino acids 1-25 of SEQ ID NO: 160);DSSPLLQFGGQVRLRHLYTSG (M6-R) (amino acids 2-22 of SEQ ID NO: 6);RPLAFSDSSPLLQFGGQVRLRHLYTSG (M7) (amino acids 1-27 of SEQ ID NO: 7);HPIPDSSPLLQWGDPIRLRHLYTSG (M8-R) (amino acids 2-26 of SEQ ID NO: 8);HPIPDSSPLLQFGWGDPIRLRHLYTSG (M9-R) (amino acids 2-28 of SEQ ID NO: 9);HPIPDSSPHVHYGWGDPIRLRHLYTSG (M10-R) (amino acids 2-28 of SEQ ID NO: 10);RPLAFSDAGPLLQWGDPIRLRHLYTSG (M11) (amino acids 1-27 of SEQ ID NO: 11);RPLAFSDAGPLLQFGWGDPIRLRHLYTSG (M12) (amino acids 1-29 of SEQ ID NO: 12);RPLAFSDAGPLLQFGGQVRLRHLYTSG (M13) (amino acids 1-27 of SEQ ID NO: 13);HPIPDSSPHVHYGGQVRLRHLYTSG (M14-R) (amino acids 2-26 of SEQ ID NO: 14);RPLAFSDAGPHVHYGGQVRLRHLYTSG (M15) (amino acids 1-27 of SEQ ID NO: 15);RPLAFSDAGPHVHWGDPIRLRHLYTSG (M16) (amino acids 1-27 of SEQ ID NO: 16);RPLAFSDAGPHVGWGDPIRLRHLYTSG (M17) (amino acids 1-27 of SEQ ID NO: 17);RPLAFSDAGPHYGWGDPIRLRHLYTSG (M18) (amino acids 1-27 of SEQ ID NO: 18);RPLAFSDAGPVYGWGDPIRLRHLYTSG (M19) (amino acids 1-27 of SEQ ID NO: 19);RPLAFSDAGPVHGWGDPIRLRHLYTSG (M20) (amino acids 1-27 of SEQ ID NO: 20);RPLAFSDAGPVHYWGDPIRLRHLYTSG (M21) (amino acids 1-27 of SEQ ID NO: 21);RPLAFSDAGPHVHGWGDPIRLRHLYTSG (M22) (amino acids 1-27 of SEQ ID NO: 22);RPLAFSDAGPHHGWGDPIRLRHLYTSG (M23) (amino acids 1-27 of SEQ ID NO: 23);RPLAFSDAGPHHYWGDPIRLRHLYTSG (M24) (amino acids 1-27 of SEQ ID NO: 24);RPLAFSDAGPHVYWGDPIRLRHLYTSG (M25) (amino acids 1-27 of SEQ ID NO: 25);RPLAFSDSSPLVHWGDPIRLRHLYTSG (M26) (amino acids 1-27 of SEQ ID NO: 26);RPLAFSDSSPHVHWGDPIRLRHLYTSG (M27) (amino acids 1-27 of SEQ ID NO: 27);RPLAFSDAGPHVWGDPIRLRHLYTSG (M28) (amino acids 1-26 of SEQ ID NO: 28);RPLAFSDAGPHVHYWGDPIRLRHLYTSG (M29) (amino acids 1-28 of SEQ ID NO: 29);RPLAFSDAGPHVHYAWGDPIRLRHLYTSG (M30) (amino acids 1-29 of SEQ ID NO: 30);RHPIPDSSPLLQFGAQVRLRHLYTSG (M31) (amino acids 1-26 of SEQ ID NO: 31);RHPIPDSSPLLQFGDQVRLRHLYTSG (M32) (amino acids 1-26 of SEQ ID NO: 32);RHPIPDSSPLLQFGPQVRLRHLYTSG (M33) (amino acids 1-26 of SEQ ID NO: 33);RHPIPDSSPLLQFGGAVRLRHLYTSG (M34) (amino acids 1-26 of SEQ ID NO: 34);RHPIPDSSPLLQFGGEVRLRHLYTSG (M35) (amino acids 1-26 of SEQ ID NO: 35);RHPIPDSSPLLQFGGNVRLRHLYTSG (M36) (amino acids 1-26 of SEQ ID NO: 36);RHPIPDSSPLLQFGGQARLRHLYTSG (M37) (amino acids 1-26 of SEQ ID NO: 37);RHPIPDSSPLLQFGGQIRLRHLYTSG (M38) (amino acids 1-26 of SEQ ID NO: 38);RHPIPDSSPLLQFGGQTRLRHLYTSG (M39) (amino acids 1-26 of SEQ ID NO: 39);RHPIPDSSPLLQFGWGQPVRLRHLYTSG (M40) (amino acids 1-28 of SEQ ID NO: 40);DAGPHVHYGWGDPIRLRHLYTSG (M74-R) (amino acids 2-24 of SEQ ID NO: 74);VHYGWGDPIRLRHLYTSG (M75-R) (amino acids 2-19 of SEQ ID NO: 75);RLRHLYTSG (M77-R) (amino acids 2-10 of SEQ ID NO: 77);RHPIPDSSPLLQFGWGDPIRLRHLYTSG (M9) (amino acids 1-28 of SEQ ID NO: 9);RHPIPDSSPLLQWGDPIRLRHLYTSG (M8) (amino acids 1-26 of SEQ ID NO: 8);RPLAFSDAGPLLQFGWGDPIRLRHLYTSG (M12) (amino acids 1-29 of SEQ ID NO: 12);RHPIPDSSPHVHYGWGDPIRLRHLYTSG (M10) (amino acids 1-28 of SEQ ID NO: 10);RPLAFSDAGPLLQFGGQVRLRHLYTSG (M13) (amino acids 1-27 of SEQ ID NO: 13);RHPIPDSSPHVHYGGQVRLRHLYTSG (M14) (amino acids 1-26 of SEQ ID NO: 14);RPLAFSDAGPHVHYGGDIRLRHLYTSG (M43) amino acids 1-27 of SEQ ID NO: 43); orRDSSPLLQFGGQVRLRHLYTSG (M6) (amino acids 1-22 of SEQ ID NO: 6).

In various further particular aspects, a peptide sequence includes orconsists of:

(SEQ ID NO: 160) HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (SEQ ID NO: 138 or 161)DSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLYNVYRSEKHRLPVSLSSAKQRQVTGLEAVRSPSFEK; (SEQ ID NO: 1 or 139)RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; (SEQ ID NO: 2 or 140)RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK; or (SEQ ID NO: 141)DSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK;or a subsequence or fragment thereof of any of the foregoing peptidesequences. In certain embodiments of any of the foregoing peptidesequences, the R terminal residue is deleted.

In various still additional particular aspects, a peptide sequenceincludes the addition of amino acid residues 30-194 of SEQ ID NO:99[FGF19] at the C-terminus, resulting in a chimeric polypeptide.

In various further embodiments, a peptide or chimeric sequence has anamino acid substitution, an addition, insertion or is a subsequence thathas at least one amino acid deleted. Such amino acid substitutions,additions, insertions and deletions of a peptide sequence can be 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues (10-20, 20-30,30-40, 40-50, etc.), for example, at the N- or C-terminus, or internal.For example, a subsequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid deletions from theamino terminus, the carboxy-terminus or internally. In a particularaspect, the amino acid substitution, or deletion is at any of amino acidpositions 8-20 of FGF19 (AGPHVHYGWGDPI) (SEQ ID NO:187).

In various still more particular aspects, a peptide or chimeric sequenceincludes all or a portion of an FGF19 sequence set forth as:PHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ ID NO:188) positioned atthe C-terminus of the peptide, or the amino terminal “R” residue isdeleted from the sequence.

In various embodiments, a peptide or chimeric sequence has a function oractivity greater or less than a comparison sequence. In particularembodiments, a peptide sequence has reduced HCC formation compared toFGF19, or an FGF 19 variant sequence having any of GQV, GDI, WGPI (SEQID NO:171), WGDPV (SEQ ID NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ IDNO:174), GPI, WGQPI (SEQ ID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQID NO:177), WADPI (SEQ ID NO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ IDNO:180), WDPI (SEQ ID NO:181), WGDI (SEQ ID NO:182), WGDP (SEQ IDNO:183) or FGDPI (SEQ ID NO:184) substituted for the WGDPI (SEQ IDNO:170) sequence at amino acids 16-20 of FGF19; or has greater glucoselowering activity compared to FGF19, or an FGF 19 variant sequencehaving any of GQV, GDI, WGPI (SEQ ID NO:171), WGDPV (SEQ ID NO:172),WGDI (SEQ ID NO:173), GDPI (SEQ ID NO:174), GPI, WGQPI (SEQ ID NO:175),WGAPI (SEQ ID NO:176), AGDPI (SEQ ID NO:177), WADPI (SEQ ID NO:178),WGDAI (SEQ ID NO:179), WGDPA (SEQ ID NO:180), WDPI (SEQ ID NO:181), WGDI(SEQ ID NO:182), WGDP (SEQ ID NO:183) or FGDPI (SEQ ID NO:184)substituted for the WGDPI (SEQ ID NO:170) sequence at amino acids 16-20of FGF19; has less lipid increasing activity compared to FGF19, or anFGF 19 variant sequence having any of GQV, GDI, WGPI (SEQ ID NO:171),WGDPV (SEQ ID NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ ID NO:174), GPI,WGQPI (SEQ ID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQ ID NO:177),WADPI (SEQ ID NO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ ID NO:180),WDPI (SEQ ID NO:181), WGDI (SEQ ID NO:182), WGDP (SEQ ID NO:183) orFGDPI (SEQ ID NO:184) substituted for the WGDPI (SEQ ID NO:170) sequenceat amino acids 16-20 of FGF19; or has less triglyceride, cholesterol,non-HDL or HDL increasing activity compared to FGF19, or an FGF 19variant sequence having any of GQV, GDI, WGPI (SEQ ID NO:171), WGDPV(SEQ ID NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ ID NO:174), GPI, WGQPI(SEQ ID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQ ID NO:177), WADPI(SEQ ID NO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ ID NO:180), WDPI (SEQID NO:181), WGDI (SEQ ID NO:182), WGDP (SEQ ID NO:183) or FGDPI (SEQ IDNO:184) substituted for the WGDPI (SEQ ID NO:170) sequence at aminoacids 16-20 of FGF19; or the peptide sequence has less lean massreducing activity compared to FGF21. Such functions and activities canbe ascertained in vitro or in vivo, for example, in a db/db mouse.

In additional various embodiments, a peptide or chimeric sequence has aneffect on function or activity of other molecules. In one aspect, apeptide sequence maintains or increases an FGFR4 mediated activity. Inanother aspect, a peptide sequence binds to fibroblast growth factorreceptor 4 (FGFR4) or activates FGFR4, or does not detectably bind toFGFR4 or activate FGFR4. In an additional aspect, a peptide sequencebinds to FGFR4 with an affinity less than, comparable to or greater thanFGF19 binding affinity for FGFR4. In a further aspect, a peptidesequence activates FGFR4 to an extent or amount less than, comparable toor greater than FGF19 activates FGFR4.

In further additional various embodiments, a peptide or chimericsequence includes one or more L-amino acids, D-amino acids,non-naturally occurring amino acids, or amino acid mimetic, derivativeor analogue. In still further various embodiments, a peptide or chimericsequence has an N-terminal region, or a C-terminal region, or a FGF19sequence portion, or an FGF21 sequence portion, joined by a linker orspacer.

In still additional embodiments, a chimeric peptide or peptide sequenceis included in a pharmaceutical composition, which in turn can be usedfor practicing the invention methods and uses. Such compositions includecombinations of inactive or other active ingredients. In one embodiment,a composition, such as a pharmaceutical composition includes chimericpeptide sequence or peptide sequence and an agent that improves bileacid homeostasis.

Uses and methods of treatment that include administration or delivery ofa chimeric peptide or peptide sequence are also provided. In particularembodiments, a use or method of treatment of a subject includesadministering an invention chimeric peptide or peptide sequence to asubject, such as a subject having, or at risk of having, a disordertreatable by an invention peptide sequence, in an amount effective fortreating the disorder. In a further embodiment, a method or use includesadministering an invention chimeric peptide or peptide sequence to asubject, such as a subject having a bile acid related or associateddisorder.

In particular aspects of the invention methods and uses, a chimericpeptide sequence or peptide sequence is administered to a subject in anamount effective to improve or provide bile acid homeostasis.Non-limiting exemplary bile acid related or associated disorderstreatable according to the invention methods and uses include: metabolicsyndrome; a lipid- or glucose-related disorder; cholesterol ortriglyceride metabolism; type 2 diabetes; cholestasis, including, forexample diseases of intrahepatic cholestasis (e.g., PBC, PFIC, PSC, PIC,neonatal cholestasis, and drug induced cholestasis (e.g., estrogen)),and diseases of extrahepatic cholestasis (e.g., bile cut compressionfrom tumor, bile duct blockade by gall stones); bile acid malabsorptionand other disorders involving the distal small intestine, includingileal resection, inflammatory bowel diseases (e.g., Crohn's disease andulcerative colitis), disorders impairing absorption of bile acids nototherwise characterized (idiopathic)) leading to diarrhea (e.g., BAD)and GI symptoms, and GI, liver, and/or biliary cancers (e.g., coloncancer and hepatocellular cancer); and/or bile acid synthesisabnormalities, such as those contributing to NASH, cirrhosis and portalhypertension. In one embodiment, the bile acid related or associateddisorder is bile acid malabsorption. In another embodiment, the bileacid related or associated disorder is diarrhea. In another embodiment,the bile acid related or associated disorder is cholestasis (e.g.,intrahepatic or extrahepatic cholestasis). In another embodiment, thebile acid related or associated disorder is primary billiary cirrhosis.In another embodiment, the bile acid related or associated disorder isprimary sclerosing cholangitis. In another embodiment, the bile acidrelated or associated disorder is PFIC (e.g., progressive PFIC).

Methods and uses of analyzing and/or identifying a chimeric peptidesequence or peptide sequence are also provided, such as chimeric peptidesequences and peptide sequences that modulate bile acid homeostasis,optionally without having substantial or significant HCC activity. Inone embodiment, a method or use includes: a) providing a candidatepeptide sequence; b) administering the candidate peptide sequence to atest animal; c) measuring bile acid levels of the animal afteradministration of the candidate peptide sequence, to determine if thecandidate peptide sequence modulates bile acid homeostasis; and d)analyzing the candidate peptide sequence for induction of HCC in theanimal, or expression of a marker correlating with HCC activity. Acandidate peptide that modulates bile acid homeostasis but does not havesubstantial HCC activity thereby identifies the candidate peptidesequence as a peptide sequence having that modulates bile acidhomeostasis without substantial HCC activity.

In a particular aspect, the chimeric peptide sequence or peptidesequence is also analyzed for induction of HCC in the animal (e.g.,assessing a hepatic tissue sample from the test animal), or expressionof a marker correlating with HCC activity. Such methods and usesidentify the candidate as having bile acid homeostasis modulatingactivity, optionally also without substantial or significant HCCactivity.

DESCRIPTION OF DRAWINGS

FIG. 1 shows cyp7a1 expression in db/db mice dosed intraperitoneallywith the indicated concentrations of FGF19 and FGF21 (SEQ ID NOs:99 and100).

FIG. 2A-2D show cyp7a1 expression in human primary hepatocytes followingdosing of A) variant M1 (SEQ ID NO:1); B) variant M2 (SEQ ID NO:2); C)variant M5 (SEQ ID NO:5); and D) variant M32 (SEQ ID NO:32).

FIG. 3A-3D show cyp7a1 expression in human primary hepatocytes followingdosing of A) variant M69 (SEQ ID NO:69); B) variant M75 (SEQ ID NO:75);C) variant M70 (SEQ ID NO:70); and D) variant M76 (SEQ ID NO:76).

FIG. 4A-4D show cyp7a1 expression in human primary hepatocytes followingdosing of A) variant M85 (SEQ ID NO:85); B) variant M96 (SEQ ID NO:96);C) variant M90 (SEQ ID NO:90); and D) variant M98 (SEQ ID NO:98).

FIG. 5 is a table showing the cyp7a1 IC₅₀ (pM), relative cyp7a1expression and HCC core of the indicated variants: M1, M2, M5, M32, M69,M70, M75, M76, M85, M90, M96 and M98.

FIG. 6 depicts the results of a human clinical trial, showingadministration of M70 is able to suppress 7a-hydroxy-4-cholsten-3-one(C4), a marker of bile acid synthesis, as compared to a placebo.

FIG. 7 depicts that the expression of FGFR4/β-klotho complex in L6 cellspotentiates activation of intracellular signaling pathways by FGF19, M3and M70.

DETAILED DESCRIPTION

The invention provides chimeric and peptide sequences that modulate bileacid homeostasis and are able to treat a bile-acid related or associateddisorder. In one embodiment, a chimeric peptide sequence includes orconsists of an N-terminal region having at least seven amino acidresidues and the N-terminal region having a first amino acid positionand a last amino acid position, where the N-terminal region has a DSSPL(SEQ ID NO:121) or DASPH (SEQ ID NO:122) sequence; and a C-terminalregion having a portion of FGF19 and the C-terminal region having afirst amino acid position and a last amino acid position, where theC-terminal region includes amino acid residues 16-29 of FGF19(WGDPIRLRHLYTSG; SEQ ID NO:169) and the W residue corresponds to thefirst amino acid position of the C-terminal region.

In another embodiment, a chimeric peptide sequence includes or consistsof an N-terminal region having a portion of FGF21 and the N-terminalregion having a first amino acid position and a last amino acidposition, where the N-terminal region has a GQV sequence and the Vresidue corresponds to the last amino acid position of the N-terminalregion; and a C-terminal region having a portion of FGF19 and theC-terminal region having a first amino acid position and a last aminoacid position where the C-terminal region includes amino acid residues21-29 of FGF19 (RLRHLYTSG; SEQ ID NO: 185) and the R residue correspondsto the first position of the C-terminal region.

In further embodiments, a peptide sequence includes or consists of aFGF19 sequence variant having one or more amino acid substitutions,insertions or deletions compared to a reference or wild type FGF19. Inadditional embodiments, a peptide sequence includes or consists of aFGF21 sequence variant having one or more amino acid substitutions,insertions or deletions compared to a reference or wild type FGF21. Inyet additional embodiments, a peptide sequence includes or consists of aportion of an FGF19 sequence fused to a portion of an FGF21 sequence. Instill additional embodiments, a peptide sequence includes or consists ofa portion of an FGF19 sequence fused to a portion of an FGF21 sequence,where the FGF19 and/or FGF21 sequence portion(s) have one or more aminoacid substitutions, insertions or deletions compared to a reference orwild type FGF19 and/or FGF21.

The invention also provides methods and uses of treating a subjecthaving or at risk of having a disorder treatable using variants andfusions of FGF19 and/or FGF21 peptide sequences. In one embodiment, amethod or use includes contacting or administering to a subject one ormore variant or fusion FGF19 and/or FGF21 peptide sequences in an amounteffective for treating a bile-acid related or associated disorder. Inanother embodiment, a method or use includes contacting or administeringto a subject one or more nucleic acid molecules encoding a variant orfusion FGF19 and/or FGF21 peptide sequence (for example, an expressioncontrol element in operable linkage with the nucleic acid encoding thepeptide sequence, optionally including a vector), in an amount effectivefor treating a bile-acid related or associated disorder.

A representative reference or wild type FGF19 sequence is set forth as:

(SEQ ID NO: 99) RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK.

A representative reference or wild type FGF21 sequence is set forth as:HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS (SEQ IDNO:100). FGF21 allelic variants include, e.g., M70, M71 and M72.

The terms “peptide,” “protein,” and “polypeptide” sequence are usedinterchangeably herein to refer to two or more amino acids, or“residues,” including chemical modifications and derivatives of aminoacids, covalently linked by an amide bond or equivalent. The amino acidsforming all or a part of a peptide may be from among the known 21naturally occurring amino acids, which are referred to by both theirsingle letter abbreviation or common three-letter abbreviation. In thepeptide sequences of the invention, conventional amino acid residueshave their conventional meaning. Thus, “Leu” is leucine, “Ile” isisoleucine, “Nle” is norleucine, and so on.

Exemplified herein are peptide sequences, distinct from reference FGF19and FGF21 polypeptides set forth herein, that modulate bile acidhomeostasis, in vivo (e.g., Tables 1-10 and the Sequence Listing).Non-limiting particular examples are a peptide sequence withamino-terminal amino acids 1-16 of FGF21 fused to carboxy-terminal aminoacids 21-194 of FGF19; a peptide sequence with amino-terminal aminoacids 1-147 of FGF19 fused to carboxy-terminal amino acids 147-181 ofFGF21; a peptide sequence with amino-terminal amino acids 1-20 of FGF19fused to carboxy-terminal amino acids 17-181 of FGF21; a peptidesequence with amino-terminal amino acids 1-146 of FGF21 fused tocarboxy-terminal amino acids 148-194 of FGF19; and a peptide sequencewith amino-terminal amino acids 1-20 of FGF19 fused to internal aminoacids 17-146 of FGF21 fused to carboxy-terminal amino acids 148-194 ofFGF19.

Additional particular peptides sequences have a WGDPI (SEQ ID NO:170)sequence motif corresponding to the WGDPI sequence of amino acids 16-20of FGF19 (SEQ ID NO:99), lack a WGDPI (SEQ ID NO:170) sequence motifcorresponding to the WGDPI sequence of amino acids 16-20 of FGF19 (SEQID NO:99), or have a substituted (i.e., mutated) WGDPI (SEQ ID NO:170)sequence motif corresponding to FGF19 WGDPI sequence of amino acids16-20 of FGF19 (SEQ ID NO:99).

Particular peptide sequences of the invention also include sequencesdistinct from FGF19 and FGF21 (e.g., as set forth herein), and FGF 19variant sequences having any GQV, GDI, WGPI (SEQ ID NO:171), WGDPV (SEQID NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ ID NO:174), GPI, WGQPI (SEQID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQ ID NO:177), WADPI (SEQ IDNO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ ID NO:180), WDPI (SEQ IDNO:181), WGDI (SEQ ID NO:182), WGDP (SEQ ID NO:183) or FGDPI (SEQ IDNO:184) substituted for FGF19 WGDPI (SEQ ID NO:170) sequence at aminoacids 16-20. Accordingly, the wild-type FGF19 and FGF21 (e.g., as setforth herein as SEQ ID NOS:99 and 100, respectively) may be excludedsequences, and FGF19 having any of GQV, GDI, WGPI (SEQ ID NO:171), WGDPV(SEQ ID NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ ID NO:174), GPI, WGQPI(SEQ ID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQ ID NO:177), WADPI(SEQ ID NO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ ID NO:180), WDPI (SEQID NO:181), WGDI (SEQ ID NO:182), WGDP (SEQ ID NO:183) or FGDPI (SEQ IDNO:184) substituted for the WGDPI (SEQ ID NO:170) sequence at aminoacids 16-20 of FGF19 may also be excluded. This exclusion, however, doesnot apply to where a sequence has, for example, 3 FGF21 residues fusedto FGF19 having, for example, any of GQV, GQV, GDI, or GPI, or 2 FGF21residues fused to any of WGPI (SEQ ID NO:171), WGDI (SEQ ID NO:173),GDPI (SEQ ID NO:174), WDPI (SEQ ID NO:181), WGDI (SEQ ID NO:182), orWGDP (SEQ ID NO:183).

Particular non-limiting examples of peptide sequences include or consistof all or a part of a sequence variant specified herein as M1-M98 (SEQID NOs:1-52, 192, and 54-98, respectively). More particular non-limitingexamples of peptide sequences include or consist of all or a part of asequence set forth as:

(SEQ ID NO: 160)HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M5-R) (FGF21 sequences can also include an “R”residue at the amino terminus); (SEQ ID NO: 138 and 161)DSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K;(SEQ ID NO: 1 or 139)RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M1); (SEQ ID NO: 2 or 140)RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M2); (SEQ ID NO: 141)DSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSF EK;(SEQ ID NO: 69)RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M69); (SEQ ID NO: 52)RDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K (M52);(SEQ ID NO: 160)HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M5-R); (SEQ ID NO: 71)HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHSLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS (M71);(SEQ ID NO: 72)HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS (M72);(SEQ ID NO: 73)HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVVQDELQGVGGEGCHMHPENCKTLLTDIDRTHTEKPVWDGITGE (M73); (SEQ ID NO: 3)RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M3); (SEQ ID NO: 48, 6 or 148)RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K (M48);(SEQ ID NO: 49, 7 or 149)RPLAFSDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M49); (SEQ ID NO: 50)RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M50); (SEQ ID NO: 51, 36 or 155)RHPIPDSSPLLQFGGNVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M51); (SEQ ID NO: 192)MDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFE K (M53);(SEQ ID NO: 70)MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M70); (SEQ ID NO: 193)RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M139); (SEQ ID NO: 194)RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIREDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M140); (SEQ ID NO: 195)RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILCDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M141);  or (SEQ ID NO: 196)RPLAFSDAGPHVHYGWGDPIRQRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M160);or a subsequence or fragment thereof of any of the foregoing peptidesequences. In certain embodiments of any of the foregoing peptidesequences, the R terminal residue is deleted.

Additional particular non-limiting examples of peptide sequences, havingat the N-terminus, a peptide sequence including or consisting of all ora part of any of:

HPIPDSSPLLQFGGQVRLRHLYTSG (M5-R) (amino acids 1-25 of SEQ ID NO: 160);DSSPLLQFGGQVRLRHLYTSG (M6) (M6-R) (amino acids 2-22 of SEQ ID NO: 6);RPLAFSDSSPLLQFGGQVRLRHLYTSG (M7) (amino acids 1-27 of SEQ ID NO: 7);HPIPDSSPLLQWGDPIRLRHLYTSG (M8-R) (amino acids 2-26 of SEQ ID NO: 8);HPIPDSSPLLQFGWGDPIRLRHLYTSG (M9-R) (amino acids 2-28 of SEQ ID NO: 9);HPIPDSSPHVHYGWGDPIRLRHLYTSG (M10-R) (amino acids 2-28 of SEQ ID NO: 10);RPLAFSDAGPLLQWGDPIRLRHLYTSG (M11) (amino acids 1-27 of SEQ ID NO: 11);RPLAFSDAGPLLQFGWGDPIRLRHLYTSG (M12) (amino acids 1-29 of SEQ ID NO: 12);RPLAFSDAGPLLQFGGQVRLRHLYTSG (M13) (amino acids 1-27 of SEQ ID NO: 13);HPIPDSSPHVHYGGQVRLRHLYTSG (M14-R) (amino acids 2-26 of SEQ ID NO: 14);RPLAFSDAGPHVHYGGQVRLRHLYTSG (M15) (amino acids 1-27 of SEQ ID NO: 15);RPLAFSDAGPHVHWGDPIRLRHLYTSG (M16) (amino acids 1-27 of SEQ ID NO: 16);RPLAFSDAGPHVGWGDPIRLRHLYTSG (M17) (amino acids 1-27 of SEQ ID NO: 17);RPLAFSDAGPHYGWGDPIRLRHLYTSG (M18) (amino acids 1-27 of SEQ ID NO: 18);RPLAFSDAGPVYGWGDPIRLRHLYTSG (M19) (amino acids 1-27 of SEQ ID NO: 19);RPLAFSDAGPVHGWGDPIRLRHLYTSG (M20) (amino acids 1-27 of SEQ ID NO: 20);RPLAFSDAGPVHYWGDPIRLRHLYTSG (M21) (amino acids 1-27 of SEQ ID NO: 21);RPLAFSDAGPHVHGWGDPIRLRHLYTSG (M22) (amino acids 1-27 of SEQ ID NO: 22);RPLAFSDAGPHHGWGDPIRLRHLYTSG (M23) (amino acids 1-27 of SEQ ID NO: 23);RPLAFSDAGPHHYWGDPIRLRHLYTSG (M24) (amino acids 1-27 of SEQ ID NO: 24);RPLAFSDAGPHVYWGDPIRLRHLYTSG (M25) (amino acids 1-27 of SEQ ID NO: 25);RPLAFSDSSPLVHWGDPIRLRHLYTSG (M26) (amino acids 1-27 of SEQ ID NO: 26);RPLAFSDSSPHVHWGDPIRLRHLYTSG (M27) (amino acids 1-27 of SEQ ID NO: 27);RPLAFSDAGPHVWGDPIRLRHLYTSG (M28) (amino acids 1-26 of SEQ ID NO: 28);RPLAFSDAGPHVHYWGDPIRLRHLYTSG (M29) (amino acids 1-28 of SEQ ID NO: 29);RPLAFSDAGPHVHYAWGDPIRLRHLYTSG (M30) (amino acids 1-29 of SEQ ID NO: 30);RHPIPDSSPLLQFGAQVRLRHLYTSG (M31) (amino acids 1-26 of SEQ ID NO: 31);RHPIPDSSPLLQFGDQVRLRHLYTSG (M32) (amino acids 1-26 of SEQ ID NO: 32);RHPIPDSSPLLQFGPQVRLRHLYTSG (M33) (amino acids 1-26 of SEQ ID NO: 33);RHPIPDSSPLLQFGGAVRLRHLYTSG (M34) (amino acids 1-26 of SEQ ID NO: 34);RHPIPDSSPLLQFGGEVRLRHLYTSG (M35) (amino acids 1-26 of SEQ ID NO: 35);RHPIPDSSPLLQFGGNVRLRHLYTSG (M36) (amino acids 1-26 of SEQ ID NO: 36);RHPIPDSSPLLQFGGQARLRHLYTSG (M37) (amino acids 1-26 of SEQ ID NO: 37);RHPIPDSSPLLQFGGQIRLRHLYTSG (M38) (amino acids 1-26 of SEQ ID NO: 38);RHPIPDSSPLLQFGGQTRLRHLYTSG (M39) (amino acids 1-26 of SEQ ID NO: 39);RHPIPDSSPLLQFGWGQPVRLRHLYTSG (M40) (amino acids 1-28 of SEQ ID NO: 40);DAGPHVHYGWGDPIRLRHLYTSG (M74-R) (amino acids 2-24 of SEQ ID NO: 74);VHYGWGDPIRLRHLYTSG (M75-R) (amino acids 2-19 of SEQ ID NO: 75);RLRHLYTSG (M77-R) (amino acids 2-10 of SEQ ID NO: 77);RHPIPDSSPLLQFGWGDPIRLRHLYTSG (M9) (amino acids 1-28 of SEQ ID NO: 9);RHPIPDSSPLLQWGDPIRLRHLYTSG (M8) (amino acids 1-26 of SEQ ID NO: 8);RPLAFSDAGPLLQFGWGDPIRLRHLYTSG (M12) (amino acids 1-29 of SEQ ID NO: 12);RHPIPDSSPHVHYGWGDPIRLRHLYTSG (M10) (amino acids 1-28 of SEQ ID NO: 10);RPLAFSDAGPLLQFGGQVRLRHLYTSG (M13) (amino acids 1-27 of SEQ ID NO: 13);RHPIPDSSPHVHYGGQVRLRHLYTSG (M14) (amino acids 1-26 of SEQ ID NO: 14);RPLAFSDAGPHVHYGGDIRLRHLYTSG (M43) amino acids 1-27 of SEQ ID NO: 43); orRDSSPLLQFGGQVRLRHLYTSG (M6) (amino acids 1-22 of SEQ ID NO: 6);and for any of the foregoing peptide sequences the amino terminal Rresidue may be deleted.

Peptide sequences of the invention additionally include those withreduced or absent induction or formation of HCC compared to FGF19, or anFGF 19 variant sequence having any of GQV, GDI, WGPI (SEQ ID NO:171),WGDPV (SEQ ID NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ ID NO:174), GPI,WGQPI (SEQ ID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQ ID NO:177),WADPI (SEQ ID NO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ ID NO:180),WDPI (SEQ ID NO:181), WGDI (SEQ ID NO:182), WGDP (SEQ ID NO:183) orFGDPI (SEQ ID NO:184) substituted for the WGDPI (SEQ ID NO:170) sequenceat amino acids 16-20 of FGF19. Peptide sequences of the invention alsoinclude those with greater glucose lowering activity compared to FGF19,or an FGF 19 variant sequence having any of GQV, GDI, WGPI, WGPI (SEQ IDNO:171), WGDPV (SEQ ID NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ IDNO:174), GPI, WGQPI (SEQ ID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQID NO:177), WADPI (SEQ ID NO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ IDNO:180), WDPI (SEQ ID NO:181), WGDI (SEQ ID NO:182), WGDP (SEQ IDNO:183) or FGDPI (SEQ ID NO:184) substituted for the WGDPI (SEQ IDNO:170) sequence at amino acids 16-20 of FGF19. Peptide sequences of theinvention moreover include those with less lipid (e.g., triglyceride,cholesterol, non-HDL or HDL) increasing activity compared to FGF19, oran FGF 19 variant sequence having any of GQV, GDI, WGPI (SEQ ID NO:171),WGDPV (SEQ ID NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ ID NO:174), GPI,WGQPI (SEQ ID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQ ID NO:177),WADPI (SEQ ID NO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ ID NO:180),WDPI (SEQ ID NO:181), WGDI (SEQ ID NO:182), WGDP (SEQ ID NO:183) orFGDPI (SEQ ID NO:184) substituted for the WGDPI (SEQ ID NO:170) sequenceat amino acids 16-20 of FGF19.

Typically, the number of amino acids or residues in an invention peptidesequence will total less than about 250 (e.g., amino acids or mimeticsthereof). In various particular embodiments, the number of residuescomprise from about 20 up to about 200 residues (e.g., amino acids ormimetics thereof). In additional embodiments, the number of residuescomprise from about 50 up to about 200 residues (e.g., amino acids ormimetics thereof). In further embodiments, the number of residuescomprise from about 100 up to about 195 residues (e.g., amino acids ormimetics thereof) in length.

Amino acids or residues can be linked by amide or by non-natural andnon-amide chemical bonds including, for example, those formed withglutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, orN,N′-dicyclohexylcarbodiimide (DCC). Non-amide bonds include, forexample, ketomethylene, aminomethylene, olefin, ether, thioether and thelike (see, e.g., Spatola in Chemistry and Biochemistry of Amino Acids,Peptides and Proteins, Vol. 7, pp 267-357 (1983), “Peptide and BackboneModifications,” Marcel Decker, NY). Thus, when a peptide of theinvention includes a portion of an FGF19 sequence and a portion of anFGF21 sequence, the two portions need not be joined to each other by anamide bond, but can be joined by any other chemical moiety or conjugatedtogether via a linker moiety.

The invention also includes subsequences, variants and modified forms ofthe exemplified peptide sequences (including the FGF19 and FGF21variants and subsequences listed in Tables 1-10 and Sequence Listing),so long as the foregoing retains at least a detectable or measurableactivity or function. For example, certain exemplified variant peptideshave FGF19 C-terminal sequence,PHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ ID NO:188) at theC-terminal portion, e.g., following the “TSG” amino acid residues of thevariant.

Also, certain exemplified variant peptides, for example, those havingall or a portion of FGF21 sequence at the amino-terminus, have an “R”residue positioned at the N-terminus, which can be omitted. Similarly,certain exemplified variant peptides, include an “M” residue positionedat the N-terminus, which can be appended to or further substituted foran omitted residue, such as an “R” residue. More particularly, invarious embodiments peptide sequences at the N-terminus include any of:RDSS (SEQ ID NO:115), DSS, MDSS (SEQ ID NO:116) or MRDSS (SEQ IDNO:117). Furthermore, in cells when a “M” residue is adjacent to a “S”residue, the “M” residue may be cleaved such that the “M” residue isdeleted from the peptide sequence, whereas when the “M” residue isadjacent to a “D” residue, the “M” residue may not be cleaved. Thus, byway of example, in various embodiments peptide sequences include thosewith the following residues at the N-terminus: MDSSPL (SEQ ID NO:119),MSDSSPL (SEQ ID NO:120) (cleaved to SDSSPL (SEQ ID NO:112)) and MSSPL(SEQ ID NO:113) (cleaved to SSPL (SEQ ID NO:114)).

Accordingly, the “peptide,” “polypeptide,” and “protein” sequences ofthe invention include subsequences, variants and modified forms of theFGF19 and FGF21 variants and subsequences listed in Tables 1-10 andSequence Listing, and the FGF19/FGF21 fusions and chimeras listed inTables 1-10 and Sequence Listing, so long as the subsequence, variant ormodified form (e.g., fusion or chimera) retains at least a detectableactivity or function, e.g., modulates bile acid homeostasis.

As used herein, the term “modify” and grammatical variations thereof,means that the composition deviates relative to a reference composition,such as a peptide sequence. Such modified peptide sequences, nucleicacids and other compositions may have greater or less activity orfunction, or have a distinct function or activity compared with areference unmodified peptide sequence, nucleic acid, or othercomposition, or may have a property desirable in a protein formulatedfor therapy (e.g. serum half-life), to elicit antibody for use in adetection assay, and/or for protein purification. For example, a peptidesequence of the invention can be modified to increase serum half-life,to increase in vitro and/or in vivo stability of the protein, etc.

Particular examples of such subsequences, variants and modified forms ofthe peptide sequences exemplified herein (e.g., a peptide sequencelisted in Tables 1-10 and Sequence Listing) include substitutions,deletions and/or insertions/additions of one or more amino acids, to orfrom the amino terminus, the carboxy-terminus or internally. One exampleis a substitution of an amino acid residue for another amino acidresidue within the peptide sequence. Another is a deletion of one ormore amino acid residues from the peptide sequence, or an insertion oraddition of one or more amino acid residues into the peptide sequence.

The number of residues substituted, deleted or inserted/added are one ormore amino acids (e.g., 1-3, 3-5, 5-10, 10-20, 20-30, 30-40, 40-50,50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140,140-150, 150-160, 160-170, 170-180, 180-190, 190-200, 200-225, 225-250,or more) of a peptide sequence. Thus, an FGF19 or FGF21 sequence canhave few or many amino acids substituted, deleted or inserted/added(e.g., 1-3, 3-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80,80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 150-160,160-170, 170-180, 180-190, 190-200, 200-225, 225-250, or more). Inaddition, an FGF19 amino acid sequence can include or consist of anamino acid sequence of about 1-3, 3-5, 5-10, 10-20, 20-30, 30-40, 40-50,50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140,140-150, 150-160, 160-170, 170-180, 180-190, 190-200, 200-225, 225-250,or more amino acids from FGF21; or an FGF21 amino acid or sequence caninclude or consist of an amino acid sequence of about 1-3, 3-5, 5-10,10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110,110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190,190-200, 200-225, 225-250, or more amino acids from FGF19.

Specific examples of substitutions include substituting a D residue foran L-residue. Accordingly, although residues are listed in the L-isomerconfiguration D-amino acids at any particular or all positions of thepeptide sequences of the invention are included, unless a D-isomer leadsto a sequence that has no detectable or measurable function.

Additional specific examples are non-conservative and conservativesubstitutions. A “conservative substitution” is a replacement of oneamino acid by a biologically, chemically or structurally similarresidue. Biologically similar means that the substitution is compatiblewith a biological activity, e.g., glucose lowering activity.Structurally similar means that the amino acids have side chains withsimilar length, such as alanine, glycine and serine, or having similarsize, or the structure of a first, second or additional peptide sequenceis maintained. Chemical similarity means that the residues have the samecharge or are both hydrophilic and hydrophobic. Particular examplesinclude the substitution of one hydrophobic residue, such as isoleucine,valine, leucine or methionine for another, or the substitution of onepolar residue for another, such as the substitution of arginine forlysine, glutamic for aspartic acids, or glutamine for asparagine, serinefor threonine, etc. Routine assays can be used to determine whether asubsequence, variant or modified form has activity, e.g., glucoselowering activity.

Particular examples of subsequences, variants and modified forms of thepeptide sequences exemplified herein (e.g., a peptide sequence listed inTables 1-10 and Sequence Listing) have 50%-60%, 60%-70%, 70%-75%,75%-80%, 80%-85%, 85%-90%, 90%-95%, or 96%, 97%, 98%, or 99% identity toa reference peptide sequence (for example, a peptide sequence in any ofTables 1-10 Sequence Listing). The term “identity” and “homology” andgrammatical variations thereof mean that two or more referenced entitiesare the same. Thus, where two amino acid sequences are identical, theyhave the identical amino acid sequence. “Areas, regions or domains ofidentity” mean that a portion of two or more referenced entities are thesame. Thus, where two amino acid sequences are identical or homologousover one or more sequence regions, they share identity in these regions.

The extent of identity between two sequences can be ascertained using acomputer program and mathematical algorithm known in the art. Suchalgorithms that calculate percent sequence identity (homology) generallyaccount for sequence gaps and mismatches over the comparison region. Forexample, a BLAST (e.g., BLAST 2.0) search algorithm (see, e.g., Altschulet al., J. Mol. Biol. 215:403 (1990), publicly available through NCBI)has exemplary search parameters as follows: Mismatch-2; gap open 5; gapextension 2. For peptide sequence comparisons, a BLASTP algorithm istypically used in combination with a scoring matrix, such as PAM100, PAM250, BLOSUM 62 or BLOSUM 50. FASTA (e.g., FASTA2 and FASTA3) and SSEARCHsequence comparison programs are also used to quantitate the extent ofidentity (Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444 (1988);Pearson, Methods Mol Biol. 132:185 (2000); and Smith et al., J. Mol.Biol. 147:195 (1981)). Programs for quantitating protein structuralsimilarity using Delaunay-based topological mapping have also beendeveloped (Bostick et al., Biochem Biophys Res Commun. 304:320 (2003)).

In the invention peptide sequences, including subsequences, variants andmodified forms of the peptide sequences exemplified herein (e.g.,sequences listed in Tables 1-10 and Sequence Listing) an “amino acid” or“residue” includes conventional alpha-amino acids as well as beta-aminoacids, alpha, alpha disubstituted amino acids and N-substituted aminoacids wherein at least one side chain is an amino acid side chain moietyas defined herein. An “amino acid” further includes N-alkyl alpha-aminoacids, wherein the N-terminus amino group has a C₁ to C₆ linear orbranched alkyl substituent. The term “amino acid” therefore includesstereoisomers and modifications of naturally occurring protein aminoacids, non-protein amino acids, post-translationally modified aminoacids (e.g., by glycosylation, phosphorylation, ester or amide cleavage,etc.), enzymatically modified or synthesized amino acids, derivatizedamino acids, constructs or structures designed to mimic amino acids,amino acids with a side chain moiety modified, derivatized fromnaturally occurring moieties, or synthetic, or not naturally occurring,etc. Modified and unusual amino acids are included in the peptidesequences of the invention (see, for example, in Synthetic Peptides: AUser's Guide; Hruby et al., Biochem. J. 268:249 (1990); and Toniolo C.,Int. J. Peptide Protein Res. 35:287 (1990)).

In addition, protecting and modifying groups of amino acids areincluded. The term “amino acid side chain moiety” as used hereinincludes any side chain of any amino acid, as the term “amino acid” isdefined herein. This therefore includes the side chain moiety innaturally occurring amino acids. It further includes side chain moietiesin modified naturally occurring amino acids as set forth herein andknown to one of skill in the art, such as side chain moieties instereoisomers and modifications of naturally occurring protein aminoacids, non-protein amino acids, post-translationally modified aminoacids, enzymatically modified or synthesized amino acids, derivatizedamino acids, constructs or structures designed to mimic amino acids,etc. For example, the side chain moiety of any amino acid disclosedherein or known to one of skill in the art is included within thedefinition.

A “derivative of an amino acid side chain moiety” is included within thedefinition of an amino acid side chain moiety. Non-limiting examples ofderivatized amino acid side chain moieties include, for example: (a)adding one or more saturated or unsaturated carbon atoms to an existingalkyl, aryl, or aralkyl chain; (b) substituting a carbon in the sidechain with another atom, preferably oxygen or nitrogen; (c) adding aterminal group to a carbon atom of the side chain, including methyl(—CH₃), methoxy (—OCH₃), nitro (—NO₂), hydroxyl (—OH), or cyano (—C═N);(d) for side chain moieties including a hydroxy, thiol or amino groups,adding a suitable hydroxy, thiol or amino protecting group; or (e) forside chain moieties including a ring structure, adding one or more ringsubstituents, including hydroxyl, halogen, alkyl, or aryl groupsattached directly or through an ether linkage. For amino groups,suitable protecting groups are known to the skilled artisan. Providedsuch derivatization provides a desired activity in the final peptidesequence (e.g., glucose lowering, improved glucose or lipid metabolism,anti-diabetic activity, absence of substantial HCC formation ortumorigenesis, absence of substantial modulation of lean or fat mass,etc.).

An “amino acid side chain moiety” includes all such derivatization, andparticular non-limiting examples include: gamma-amino butyric acid,12-amino dodecanoic acid, alpha-aminoisobutyric acid, 6-amino hexanoicacid, 4-(aminomethyl)-cyclohexane carboxylic acid, 8-amino octanoicacid, biphenylalanine, Boc-t-butoxycarbonyl, benzyl, benzoyl,citrulline, diaminobutyric acid, pyrrollysine, diaminopropionic acid,3,3-diphenylalanine, orthonine, citrulline,1,3-dihydro-2H-isoindolecarboxylic acid, ethyl,Fmoc-fluorenylmethoxycarbonyl, heptanoyl (CH₃—(CH₂)₅—C(═O)—), hexanoyl(CH₃—(CH₂)₄—C(═O)—), homoarginine, homocysteine, homolysine,homophenylalanine, homoserine, methyl, methionine sulfoxide, methioninesulfone, norvaline (NVA), phenylglycine, propyl, isopropyl, sarcosine(SAR), tert-butylalanine, and benzyloxycarbonyl.

A single amino acid, including stereoisomers and modifications ofnaturally occurring protein amino acids, non-protein amino acids,post-translationally modified amino acids, enzymatically synthesizedamino acids, non-naturally occurring amino acids including derivatizedamino acids, an alpha, alpha disubstituted amino acid derived from anyof the foregoing (i.e., an alpha, alpha disubstituted amino acid,wherein at least one side chain is the same as that of the residue fromwhich it is derived), a beta-amino acid derived from any of theforegoing (i.e., a beta-amino acid which other than for the presence ofa beta-carbon is otherwise the same as the residue from which it isderived) etc., including all of the foregoing can be referred to hereinas a “residue.” Suitable substituents, in addition to the side chainmoiety of the alpha-amino acid, include C1 to C6 linear or branchedalkyl. Aib is an example of an alpha, alpha disubstituted amino acid.While alpha, alpha disubstituted amino acids can be referred to usingconventional L- and D-isomeric references, it is to be understood thatsuch references are for convenience, and that where the substituents atthe alpha-position are different, such amino acid can interchangeably bereferred to as an alpha, alpha disubstituted amino acid derived from theL- or D-isomer, as appropriate, of a residue with the designated aminoacid side chain moiety. Thus (S)-2-Amino-2-methyl-hexanoic acid can bereferred to as either an alpha, alpha disubstituted amino acid derivedfrom L-Nle (norleucine) or as an alpha, alpha disubstituted amino acidderived from D-Ala. Similarly, Aib can be referred to as an alpha, alphadisubstituted amino acid derived from Ala. Whenever an alpha, alphadisubstituted amino acid is provided, it is to be understood asincluding all (R) and (S) configurations thereof.

An “N-substituted amino acid” includes any amino acid wherein an aminoacid side chain moiety is covalently bonded to the backbone amino group,optionally where there are no substituents other than H in thealpha-carbon position. Sarcosine is an example of an N-substituted aminoacid. By way of example, sarcosine can be referred to as anN-substituted amino acid derivative of Ala, in that the amino acid sidechain moiety of sarcosine and Ala is the same, i.e., methyl.

Covalent modifications of the invention peptide sequences, includingsubsequences, variants and modified forms of the peptide sequencesexemplified herein (e.g., sequences listed in Tables 1-10 and SequenceListing), are included in the invention. One type of covalentmodification includes reacting targeted amino acid residues with anorganic derivatizing agent that is capable of reacting with selectedside chains or the N- or C-terminal residues of the peptide.Derivatization with bifunctional agents is useful, for instance, forcross linking peptide to a water-insoluble support matrix or surface foruse in the method for purifying anti-peptide antibodies, and vice-versa.Commonly used cross linking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of thealpha-amino groups of lysine, arginine, and histidine side chains (T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminalamine, amidation of any C-terminal carboxyl group, etc.

Exemplified peptide sequences, and subsequences, variants and modifiedforms of the peptide sequences exemplified herein (e.g., sequenceslisted in Tables 1-10 and Sequence Listing), can also includealterations of the backbone for stability, derivatives, andpeptidomimetics. The term “peptidomimetic” includes a molecule that is amimic of a residue (referred to as a “mimetic”), including but notlimited to piperazine core molecules, keto-piperazine core molecules anddiazepine core molecules. Unless otherwise specified, an amino acidmimetic of an invention peptide sequence includes both a carboxyl groupand amino group, and a group corresponding to an amino acid side chain,or in the case of a mimetic of Glycine, no side chain other thanhydrogen.

By way of example, these would include compounds that mimic the sterics,surface charge distribution, polarity, etc. of a naturally occurringamino acid, but need not be an amino acid, which would impart stabilityin the biological system. For example, Proline may be substituted byother lactams or lactones of suitable size and substitution; Leucine maybe substituted by an alkyl ketone, N-substituted amide, as well asvariations in amino acid side chain length using alkyl, alkenyl or othersubstituents, others may be apparent to the skilled artisan. Theessential element of making such substitutions is to provide a moleculeof roughly the same size and charge and configuration as the residueused to design the molecule. Refinement of these modifications will bemade by analyzing the compounds in a functional (e.g., glucose lowering)or other assay, and comparing the structure activity relationship. Suchmethods are within the scope of the skilled artisan working in medicinalchemistry and drug development.

Another type of modification of the invention peptide sequences,including subsequences, sequence variants and modified forms of theexemplified peptide sequences (including the peptides listed in Tables1-10 and Sequence Listing), is glycosylation. As used herein,“glycosylation” broadly refers to the presence, addition or attachmentof one or more sugar (e.g., carbohydrate) moieties to proteins, lipidsor other organic molecules. The use of the term “deglycosylation” hereinis generally intended to mean the removal or deletion, of one or moresugar (e.g., carbohydrate) moieties. In addition, the phrase includesqualitative changes in the glycosylation of the native proteinsinvolving a change in the type and proportions (amount) of the varioussugar (e.g., carbohydrate) moieties present.

Glycosylation can be achieved by modification of an amino acid residue,or by adding one or more glycosylation sites that may or may not bepresent in the native sequence. For example, a typicallynon-glycosylated residue can be substituted for a residue that may beglycosylated. Addition of glycosylation sites can be accomplished byaltering the amino acid sequence. The alteration to the peptide sequencemay be made, for example, by the addition of, or substitution by, one ormore serine or threonine residues (for O-linked glycosylation sites) orasparagine residues (for N-linked glycosylation sites). The structuresof N-linked and O-linked oligosaccharides and the sugar residues foundin each type may be different. One type of sugar that is commonly foundon both is N-acetylneuraminic acid (hereafter referred to as sialicacid). Sialic acid is usually the terminal residue of both N-linked andO-linked oligosaccharides and, by virtue of its negative charge, mayconfer acidic properties to the glycoprotein.

Peptide sequences of the invention may optionally be altered throughchanges at the nucleotide (e.g., DNA) level, particularly by mutatingthe DNA encoding the peptide at preselected bases such that codons aregenerated that will translate into the desired amino acids. Anothermeans of increasing the number of carbohydrate moieties on the peptideis by chemical or enzymatic coupling of glycosides to the polypeptide(see, for example, in WO 87/05330). De-glycosylation can be accomplishedby removing the underlying glycosylation site, by deleting theglycosylation by chemical and/or enzymatic means, or by substitution ofcodons encoding amino acid residues that are glycosylated. Chemicaldeglycosylation techniques are known, and enzymatic cleavage ofcarbohydrate moieties on polypeptides can be achieved by the use of avariety of endo- and exo-glycosidases.

Various cell lines can be used to produce proteins that areglycosylated. One non-limiting example is Dihydrofolate reductase(DHFR)—deficient Chinese Hamster Ovary (CHO) cells, which are a commonlyused host cell for the production of recombinant glycoproteins. Thesecells do not express the enzyme beta-galactosidealpha-2,6-sialyltransferase and therefore do not add sialic acid in thealpha-2,6 linkage to N-linked oligosaccharides of glycoproteins producedin these cells.

Another type of modification is to conjugate (e.g., link) one or moreadditional components or molecules at the N- and/or C-terminus of aninvention peptide sequence, such as another protein (e.g., a proteinhaving an amino acid sequence heterologous to the subject protein), or acarrier molecule. Thus, an exemplary peptide sequence can be a conjugatewith another component or molecule.

In certain embodiments, the amino- or carboxy-terminus of an inventionpeptide sequence can be fused with an immunoglobulin Fc region (e.g.,human Fc) to form a fusion conjugate (or fusion molecule). Fc fusionconjugates can increase the systemic half-life of biopharmaceuticals,and thus the biopharmaceutical product may have prolonged activity orrequire less frequent administration. Fc binds to the neonatal Fcreceptor (FcRn) in endothelial cells that line the blood vessels, and,upon binding, the Fc fusion molecule is protected from degradation andre-released into the circulation, keeping the molecule in circulationlonger. This Fc binding is believed to be the mechanism by whichendogenous IgG retains its long plasma half-life. Well-known andvalidated Fc-fusion drugs consist of two copies of a biopharmaceuticallinked to the Fc region of an antibody to improve pharmacokinetics,solubility, and production efficiency. More recent Fc-fusion technologylinks a single copy of a biopharmaceutical to Fc region of an antibodyto optimize the pharmacokinetic and pharmacodynamic properties of thebiopharmaceutical as compared to traditional Fc-fusion conjugates.

A conjugate modification can be used to produce a peptide sequence thatretains activity with an additional or complementary function oractivity of the second molecule. For example, a peptide sequence may beconjugated to a molecule, e.g., to facilitate solubility, storage, invivo or shelf half-life or stability, reduction in immunogenicity,delayed or controlled release in vivo, etc. Other functions oractivities include a conjugate that reduces toxicity relative to anunconjugated peptide sequence, a conjugate that targets a type of cellor organ more efficiently than an unconjugated peptide sequence, or adrug to further counter the causes or effects associated with a disorderor disease as set forth herein (e.g., diabetes).

Clinical effectiveness of protein therapeutics may be limited by shortplasma half-life and susceptibility to degradation. Studies of varioustherapeutic proteins have shown that various modifications, includingconjugating or linking the peptide sequence to any of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol (PEG),polypropylene glycol, or polyoxyalkylenes (see, for example, typicallyvia a linking moiety covalently bound to both the protein and thenonproteinaceous polymer (e.g., a PEG) can prolong half-life. SuchPEG-conjugated biomolecules have been shown to possess clinically usefulproperties, including better physical and thermal stability, protectionagainst susceptibility to enzymatic degradation, increased solubility,longer in vivo circulating half-life and decreased clearance, reducedimmunogenicity and antigenicity, and reduced toxicity.

PEGs suitable for conjugation to an invention peptide sequence isgenerally soluble in water at room temperature, and have the generalformula R(O—CH₂—CH₂)_(n)O—R, where R is hydrogen or a protective groupsuch as an alkyl or an alkanol group, and where n is an integer from 1to 1000. When R is a protective group, it generally has from 1 to 8carbons. The PEG conjugated to the peptide sequence can be linear orbranched. Branched PEG derivatives, “star-PEGs” and multi-armed PEGs areincluded in the invention. A molecular weight of the PEG used in theinvention is not restricted to any particular range, but certainembodiments have a molecular weight between 500 and 20,000 while otherembodiments have a molecular weight between 4,000 and 10,000.

The invention includes compositions of conjugates wherein the PEGs havedifferent “n” values and thus the various different PEGs are present inspecific ratios. For example, some compositions comprise a mixture ofconjugates where n=1, 2, 3 and 4. In some compositions, the percentageof conjugates where n=1 is 18-25%, the percentage of conjugates wheren=2 is 50-66%, the percentage of conjugates where n=3 is 12-16%, and thepercentage of conjugates where n=4 is up to 5%. Such compositions can beproduced by reaction conditions and purification methods know in theart.

PEG may directly or indirectly (e.g., through an intermediate) bind tothe peptide sequences of the invention. For example, in one embodiment,PEG binds via a terminal reactive group (a “spacer”). The spacer, is,for example, a terminal reactive group which mediates a bond between thefree amino or carboxyl groups of one or more of the peptide sequencesand polyethylene glycol. The PEG having the spacer which may be bound tothe free amino group includes N-hydroxysuccinylimide polyethylene glycolwhich may be prepared by activating succinic acid ester of polyethyleneglycol with N-hydroxysuccinylimide. Another activated polyethyleneglycol which may be bound to free amino group is2,4-bis(O-methoxypolyethyleneglycol)-6-chloro-s-triazine which may beprepared by reacting polyethylene glycol monomethyl ether with cyanuricchloride. The activated polyethylene glycol which is bound to the freecarboxyl group includes polyoxyethylenediamine.

Conjugation of one or more of invention peptide sequences to PEG havinga spacer may be carried out by various conventional methods. Forexample, the conjugation reaction can be carried out in solution at a pHof from 5 to 10, at temperature from 4° C. to room temperature, for 30minutes to 20 hours, utilizing a molar ratio of reagent to protein offrom 4:1 to 30:1. Reaction conditions may be selected to direct thereaction towards producing predominantly a desired degree ofsubstitution. In general, low temperature, low pH (e.g., pH=5), andshort reaction time tend to decrease the number of PEGs attached,whereas high temperature, neutral to high pH (e.g., pH ≥7), and longerreaction time tend to increase the number of PEGs attached. Variousmethods known in the art may be used to terminate the reaction. In someembodiments the reaction is terminated by acidifying the reactionmixture and freezing at, e.g., −20° C.

Invention peptide sequences including subsequences, sequence variantsand modified forms of the exemplified peptide sequences (including thepeptides listed in Tables 1-10 and Sequence Listing), further includeconjugation to large, slowly metabolized macromolecules such asproteins; polysaccharides, such as sepharose, agarose, cellulose,cellulose beads; polymeric amino acids such as polyglutamic acid,polylysine; amino acid copolymers; inactivated virus particles;inactivated bacterial toxins such as toxoid from diphtheria, tetanus,cholera, leukotoxin molecules; inactivated bacteria; and dendriticcells. Such conjugated forms, if desired, can be used to produceantibodies against peptide sequences of the invention.

Additional suitable components and molecules for conjugation include,for example, thyroglobulin; albumins such as human serum albumin (HSA);tetanus toxoid; Diphtheria toxoid; polyamino acids such aspoly(D-lysine:D-glutamic acid); VP6 polypeptides of rotaviruses;influenza virus hemagglutinin, influenza virus nucleoprotein; KeyholeLimpet Hemocyanin (KLH); and hepatitis B virus core protein and surfaceantigen; or any combination of the foregoing.

Fusion of albumin to an invention peptide sequence can, for example, beachieved by genetic manipulation, such that the DNA coding for HSA(human serum albumin), or a fragment thereof, is joined to the DNAcoding for a peptide sequence. Thereafter, a suitable host can betransformed or transfected with the fused nucleotide sequence in theform of, for example, a suitable plasmid, so as to express a fusionpolypeptide. The expression may be effected in vitro from, for example,prokaryotic or eukaryotic cells, or in vivo from, for example, atransgenic organism. In some embodiments of the invention, theexpression of the fusion protein is performed in mammalian cell lines,for example, CHO cell lines.

Further means for genetically fusing target proteins or peptides toalbumin include a technology known as Albufuse® (Novozymes BiopharmaA/S; Denmark), and the conjugated therapeutic peptide sequencesfrequently become much more effective with better uptake in the body.The technology has been utilized commercially to produce Albuferon®(Human Genome Sciences), a combination of albumin and interferon α-2Bused to treat hepatitis C infection.

Another embodiment entails the use of one or more human domainantibodies (dAb). dAbs are the smallest functional binding units ofhuman antibodies (IgGs) and have favorable stability and solubilitycharacteristics. The technology entails a dAb(s) conjugated to HSA(thereby forming a “AlbudAb”; see, e.g., EP1517921B, WO2005/118642 andWO2006/051288) and a molecule of interest (e.g., a peptide sequence ofthe invention). AlbudAbs are often smaller and easier to manufacture inmicrobial expression systems, such as bacteria or yeast, than currenttechnologies used for extending the serum half-life of peptides. As HSAhas a half-life of about three weeks, the resulting conjugated moleculeimproves the half-life. Use of the dAb technology may also enhance theefficacy of the molecule of interest.

Additional suitable components and molecules for conjugation includethose suitable for isolation or purification. Particular non-limitingexamples include binding molecules, such as biotin (biotin-avidinspecific binding pair), an antibody, a receptor, a ligand, a lectin, ormolecules that comprise a solid support, including, for example, plasticor polystyrene beads, plates or beads, magnetic beads, test strips, andmembranes.

Purification methods such as cation exchange chromatography may be usedto separate conjugates by charge difference, which effectively separatesconjugates into their various molecular weights. For example, the cationexchange column can be loaded and then washed with ˜20 mM sodiumacetate, pH ˜4, and then eluted with a linear (0 M to 0.5 M) NaClgradient buffered at a pH from 3 to 5.5, preferably at pH ˜4.5. Thecontent of the fractions obtained by cation exchange chromatography maybe identified by molecular weight using conventional methods, forexample, mass spectroscopy, SDS-PAGE, or other known methods forseparating molecular entities by molecular weight. A fraction is thenaccordingly identified which contains the conjugate having the desirednumber of PEGs attached, purified free from unmodified protein sequencesand from conjugates having other numbers of PEGs attached.

In still other embodiments, an invention peptide sequence is linked to achemical agent (e.g., an immunotoxin or chemotherapeutic agent),including, but are not limited to, a cytotoxic agent, including taxol,cytochalasin B, gramicidin D, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, andanalogs or homologs thereof. Other chemical agents include, for example,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine); alkylating agents (e.g.,mechlorethamine, carmustine and lomustine, cyclothosphamide, busulfan,dibromomannitol, streptozotocin, mitomycin C, and cisplatin);antibiotics (e.g., bleomycin); and anti-mitotic agents (e.g.,vincristine and vinblastine). Cytotoxins can be conjugated to a peptideof the invention using linker technology known in the art and describedherein.

Further suitable components and molecules for conjugation include thosesuitable for detection in an assay. Particular non-limiting examplesinclude detectable labels, such as a radioisotope (e.g., ¹²⁵I; ³⁵S, ³²P,³³P), an enzyme which generates a detectable product (e.g., luciferase,β-galactosidase, horse radish peroxidase and alkaline phosphatase), afluorescent protein, a chromogenic protein, dye (e.g., fluoresceinisothiocyanate); fluorescence emitting metals (e.g., ¹⁵²Eu);chemiluminescent compounds (e.g., luminol and acridinium salts);bioluminescent compounds (e.g., luciferin); and fluorescent proteins.Indirect labels include labeled or detectable antibodies that bind to apeptide sequence, where the antibody may be detected.

In certain embodiments, a peptide sequence of the invention isconjugated to a radioactive isotope to generate a cytotoxicradiopharmaceutical (radioimmunoconjugates) useful as a diagnostic ortherapeutic agent. Examples of such radioactive isotopes include, butare not limited to, iodine¹³¹, indium¹¹¹, yttrium⁹⁰ and lutetium¹⁷⁷.Methods for preparing radioimmunoconjugates are known to the skilledartisan. Examples of radioimmunoconjugates that are commerciallyavailable include ibritumomab, tiuxetan, and tositumomab.

Other means and methods included in the invention for prolonging thecirculation half-life, increasing stability, reducing clearance, oraltering immunogenicity or allergenicity of a peptide sequence of theinvention involves modification of the peptide sequence by hesylation,which utilizes hydroxyethyl starch derivatives linked to other moleculesin order to modify the molecule's characteristics. Various aspects ofhesylation are described in, for example, U.S. Patent Appln. Nos.2007/0134197 and 2006/0258607.

Any of the foregoing components and molecules used to modify peptidesequences of the invention, may optionally be conjugated via a linker.Suitable linkers include “flexible linkers” which are generally ofsufficient length to permit some movement between the modified peptidesequences and the linked components and molecules. The linker moleculesare generally about 6-50 atoms long. The linker molecules may also be,for example, aryl acetylene, ethylene glycol oligomers containing 2-10monomer units, diamines, diacids, amino acids, or combinations thereof.Suitable linkers can be readily selected and can be of any suitablelength, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30-50 aminoacids (e.g., Gly).

Exemplary flexible linkers include glycine polymers (G)n, glycine-serinepolymers (for example, (GS)_(n), GSGGS_(n) (SEQ ID NO:129) and GGGS_(n)(SEQ ID NO:130), where n is an integer of at least one), glycine-alaninepolymers, alanine-serine polymers, and other flexible linkers. Glycineand glycine-serine polymers are relatively unstructured, and thereforemay serve as a neutral tether between components. Exemplary flexiblelinkers include, but are not limited to GGSG (SEQ ID NO:131), GGSGG (SEQID NO:132), GSGSG (SEQ ID NO:133), GSGGG (SEQ ID NO:134), GGGSG (SEQ IDNO:189), and GSSSG (SEQ ID NO:135).

Peptide sequences of the invention, including the FGF19 and FGF21variants and subsequences and the FGF19/FGF21 fusions and chimeraslisted in Tables 1-10 and Sequence Listing, as well as subsequences,sequence variants and modified forms of the sequences listed in Tables1-10 and Sequence Listing have one or more activities as set forthherein. One example of an activity is modulating bile acid homeostasis.Another example of an activity is reduced stimulation or formation ofHCC, for example, as compared to FGF19. An additional example of anactivity is lower or reduced lipid (e.g., triglyceride, cholesterol,non-HDL) or HDL increasing activity, for example, as compared to FGF21.A further example of an activity is a lower or reduced lean muscle massreducing activity, for example, as compared to FGF21. Yet anotherexample of an activity is binding to FGFR4, or activating FGFR4, forexample, peptide sequences that bind to FGFR4 with an affinitycomparable to or greater than FGF19 binding affinity for FGFR4; andpeptide sequences that activate FGFR4 to an extent or amount comparableto or greater than FGF19 activates FGFR4. Still further examples ofactivities include treating a bile-acid related or associated disorder.

More particularly, peptide sequences of the invention, including theFGF19 and FGF21 variants and subsequences and the FGF19/FGF21 fusionsand chimeras listed in Tables 1-10 and Sequence Listing, as well assubsequences, variants and modified forms of the sequences listed inTables 1-10 and Sequence Listing include those with the followingactivities: peptide sequences modulating bile acid homeostasis ortreating a bile-acid related or associated disorder while having reducedHCC formation compared to FGF19, or an FGF 19 variant sequence havingany of GQV, GDI, WGPI (SEQ ID NO:171), WGDPV (SEQ ID NO:172), WGDI (SEQID NO:173), GDPI (SEQ ID NO:174), GPI, WGQPI (SEQ ID NO:175), WGAPI (SEQID NO:176), AGDPI (SEQ ID NO:177), WADPI (SEQ ID NO:178), WGDAI (SEQ IDNO:179), WGDPA (SEQ ID NO:180), WDPI (SEQ ID NO:181), WGDI (SEQ IDNO:182), WGDP (SEQ ID NO:183) or FGDPI (SEQ ID NO:184) substituted forthe WGDPI (SEQ ID NO:170) sequence at amino acids 16-20 of FGF19;peptide sequences having greater bile acid modulating activity comparedto FGF19, or FGF 19 variant sequence having any of GQV, GDI, WGPI (SEQID NO:171), WGDPV (SEQ ID NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ IDNO:174), GPI, WGQPI (SEQ ID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQID NO:177), WADPI (SEQ ID NO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ IDNO:180), WDPI (SEQ ID NO:181), WGDI (SEQ ID NO:182), WGDP (SEQ IDNO:183) or FGDPI (SEQ ID NO:184) substituted for the WGDPI (SEQ IDNO:170) sequence at amino acids 16-20 of FGF19; peptide sequences havingless lipid increasing activity (e.g., less triglyceride, cholesterol,non-HDL) or more HDL increasing activity compared to FGF19, or an FGF 19variant sequence having any of GQV, GDI, WGPI (SEQ ID NO:171), WGDPV(SEQ ID NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ ID NO:174), GPI, WGQPI(SEQ ID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQ ID NO:177), WADPI(SEQ ID NO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ ID NO:180), WDPI (SEQID NO:181), WGDI (SEQ ID NO:182), WGDP (SEQ ID NO:183) or FGDPI (SEQ IDNO:184) substituted for the WGDPI (SEQ ID NO:170) sequence at aminoacids 16-20 of FGF19; and peptide sequences having less lean massreducing activity as compared to FGF21.

More particularly, peptide sequences of the invention, including theFGF19 and FGF21 variants and subsequences and the FGF19/FGF21 fusionsand chimeras listed in Tables 1-10 and Sequence Listing, as well assubsequences, variants and modified forms of the sequences listed inTables 1-10 and the Sequence Listing include those with the followingactivities: peptide sequences that modulate bile acid homeostasis;peptide sequences that treat a bile-acid related or associated disorder,peptide sequences that bind to FGFR4, or activate FGFR4, such as peptidesequences that bind to FGFR4 with an affinity comparable to or greaterthan FGF19 binding affinity for FGFR4; peptide sequences that activateFGFR4 to an extent or amount comparable to or greater than FGF19activates FGFR4; peptide sequences that down-regulate or reducealdo-keto reductase gene expression, for example, compared to FGF19; andpeptide sequences that up-regulate or increase solute carrier family 1,member 2 (Slc1a2) gene expression as compared to FGF21.

As disclosed herein, variants include various N-terminal modificationsand/or truncations of FGF19, including variants in which there has beena substitution of one or several N-terminal FGF19 amino acids with aminoacids from FGF21. Such variants include variants having glucose loweringactivity, as well as a favorable lipid profile and are not measurably ordetectably tumorigenic.

In various particular aspects, modifications to the Loop-8 region ofFGF19 (residues 127-129 are defined as constituting the Loop-8 region)are disclosed herein that have glucose lowering activity and alsopossess favorable metabolic parameters without exhibiting substantialtumorigenicity. Herein, FGF19 residues 127-129 are defined asconstituting the Loop-8 region, although in the literature the Loop-8region is sometimes defined as including or consisting of other residues(e.g., residues 125-129). As set forth in Examples 8 and 9, certaincombinations of R127L and P128E substitutions to the FGF19 framework hadan unexpectedly positive effect on HCC formation. Even moresurprisingly, a combination of R127L and P128E substitutions and asubstitution of Gln (Q) for Leu (L) in the FGF19 core region (see, e.g.,core region sequence denoted in Tables 1-4, 9 and 10) had an even moresignificant effect on preventing HCC formation. Accordingly, variants ofFGF19 Loop-8 region are included since they can reduce or eliminatesubstantial, measurable or detectable HCC formation. Furthermore, theeffect of reducing HCC formation may be enhanced by modifications toamino acid residues outside of the Loop 8 region (e.g., substitutions ofamino acid residues in the core region).

Activities such as, for example, modulation of bile acid homeostasis,glucose lowering activity, analysis of a bile-acid related or associateddisorder, HCC formation or tumorigenesis, lipid increasing activity, orlean mass reducing activity can be ascertained in an animal, such as adb/db mouse. Measurement of binding to FGFR4 or activation of FGFR4 canbe ascertained by assays disclosed herein or known to the skilledartisan.

The term “bind,” or “binding,” when used in reference to a peptidesequence, means that the peptide sequence interacts at the molecularlevel. Thus, a peptide sequence that binds to FGFR4 binds to all or apart of the FGFR4 sequence. Specific and selective binding can bedistinguished from non-specific binding using assays known in the art(e.g., competition binding, immunoprecipitation, ELISA, flow cytometry,Western blotting).

Peptides and peptidomimetics can be produced and isolated using methodsknown in the art. Peptides can be synthesized, in whole or in part,using chemical methods (see, e.g., Caruthers (1980). Nucleic Acids Res.Symp. Ser. 215; Horn (1980); and Banga, A. K., Therapeutic Peptides andProteins, Formulation, Processing and Delivery Systems (1995) TechnomicPublishing Co., Lancaster, Pa.). Peptide synthesis can be performedusing various solid-phase techniques (see, e.g., Roberge Science 269:202(1995); Merrifield, Methods Enzymol. 289:3 (1997)) and automatedsynthesis may be achieved, e.g., using the ABI 431A Peptide Synthesizer(Perkin Elmer) in accordance with the manufacturer's instructions.Peptides and peptide mimetics can also be synthesized usingcombinatorial methodologies. Synthetic residues and polypeptidesincorporating mimetics can be synthesized using a variety of proceduresand methodologies known in the art (see, e.g., Organic SynthesesCollective Volumes, Gilman, et al. (Eds) John Wiley & Sons, Inc., NY).Modified peptides can be produced by chemical modification methods (see,for example, Belousov, Nucleic Acids Res. 25:3440 (1997); Frenkel, FreeRadic. Biol. Med. 19:373 (1995); and Blommers, Biochemistry 33:7886(1994)). Peptide sequence variations, derivatives, substitutions andmodifications can also be made using methods such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR based mutagenesis. Site-directed mutagenesis (Carter et al.,Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res.10:6487 (1987)), cassette mutagenesis (Wells et al., Gene 34:315(1985)), restriction selection mutagenesis (Wells et al., Philos. Trans.R. Soc. London SerA 317:415 (1986)) and other techniques can beperformed on cloned DNA to produce invention peptide sequences,variants, fusions and chimeras, and variations, derivatives,substitutions and modifications thereof.

A “synthesized” or “manufactured” peptide sequence is a peptide made byany method involving manipulation by the hand of man. Such methodsinclude but are not limited to the aforementioned, such as chemicalsynthesis, recombinant DNA technology, biochemical or enzymaticfragmentation of larger molecules, and combinations of the foregoing.

Peptide sequences of the invention including subsequences, sequencevariants and modified forms of the exemplified peptide sequences (e.g.,sequences listed in Tables 1-10 and the Sequence Listing), can also bemodified to form a chimeric molecule. In accordance with the invention,there are provided peptide sequences that include a heterologous domain.Such domains can be added to the amino-terminus or at thecarboxyl-terminus of the peptide sequence. Heterologous domains can alsobe positioned within the peptide sequence, and/or alternatively flankedby FGF19 and/or FGF21 derived amino acid sequences.

The term “peptide” also includes dimers or multimers (oligomers) ofpeptides. In accordance with the invention, there are also provideddimers or multimers (oligomers) of the exemplified peptide sequences aswell as subsequences, variants and modified forms of the exemplifiedpeptide sequences (e.g., sequences listed in Tables 1-10 and theSequence Listing).

The invention further provides nucleic acid molecules encoding peptidesequences of the invention, including subsequences, sequence variantsand modified forms of the sequences listed in Tables 1-10 and theSequence Listing, and vectors that include nucleic acid that encodes thepeptide. Accordingly, “nucleic acids” include those that encode theexemplified peptide sequences disclosed herein, as well as thoseencoding functional subsequences, sequence variants and modified formsof the exemplified peptide sequences, so long as the foregoing retain atleast detectable or measurable activity or function. For example, asubsequence, a variant or modified form of an exemplified peptidesequence disclosed herein (e.g., a sequence listed in Tables 1-10 andthe Sequence Listing) that retains some ability to lower or reduceglucose, provide normal glucose homeostasis, or reduce thehistopathological conditions associated with chronic or acutehyperglycemia in vivo, etc.

Nucleic acid, which can also be referred to herein as a gene,polynucleotide, nucleotide sequence, primer, oligonucleotide or proberefers to natural or modified purine- and pyrimidine-containing polymersof any length, either polyribonucleotides or polydeoxyribonucleotides ormixed polyribo-polydeoxyribo nucleotides and α-anomeric forms thereof.The two or more purine- and pyrimidine-containing polymers are typicallylinked by a phosphoester bond or analog thereof. The terms can be usedinterchangeably to refer to all forms of nucleic acid, includingdeoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The nucleicacids can be single strand, double, or triplex, linear or circular.Nucleic acids include genomic DNA and cDNA. RNA nucleic acid can bespliced or unspliced mRNA, rRNA, tRNA or antisense. Nucleic acidsinclude naturally occurring, synthetic, as well as nucleotide analoguesand derivatives.

As a result of the degeneracy of the genetic code, nucleic acidmolecules include sequences degenerate with respect to nucleic acidmolecules encoding the peptide sequences of the invention. Thus,degenerate nucleic acid sequences encoding peptide sequences, includingsubsequences, variants and modified forms of the peptide sequencesexemplified herein (e.g., sequences listed in Tables 1-10 and theSequence Listing), are provided. The term “complementary,” when used inreference to a nucleic acid sequence, means the referenced regions are100% complementary, i.e., exhibit 100% base pairing with no mismatches.

Nucleic acid can be produced using any of a variety of known standardcloning and chemical synthesis methods, and can be altered intentionallyby site-directed mutagenesis or other recombinant techniques known toone skilled in the art. Purity of polynucleotides can be determinedthrough sequencing, gel electrophoresis, UV spectrometry.

Nucleic acids may be inserted into a nucleic acid construct in whichexpression of the nucleic acid is influenced or regulated by an“expression control element,” referred to herein as an “expressioncassette.” The term “expression control element” refers to one or morenucleic acid sequence elements that regulate or influence expression ofa nucleic acid sequence to which it is operatively linked. An expressioncontrol element can include, as appropriate, promoters, enhancers,transcription terminators, gene silencers, a start codon (e.g., ATG) infront of a protein-encoding gene, etc.

An expression control element operatively linked to a nucleic acidsequence controls transcription and, as appropriate, translation of thenucleic acid sequence. The term “operatively linked” refers to ajuxtaposition wherein the referenced components are in a relationshippermitting them to function in their intended manner. Typically,expression control elements are juxtaposed at the 5′ or the 3′ ends ofthe genes but can also be intronic.

Expression control elements include elements that activate transcriptionconstitutively, that are inducible (i.e., require an external signal orstimuli for activation), or derepressible (i.e., require a signal toturn transcription off; when the signal is no longer present,transcription is activated or “derepressed”). Also included in theexpression cassettes of the invention are control elements sufficient torender gene expression controllable for specific cell-types or tissues(i.e., tissue-specific control elements). Typically, such elements arelocated upstream or downstream (i.e., 5′ and 3′) of the coding sequence.Promoters are generally positioned 5′ of the coding sequence. Promoters,produced by recombinant DNA or synthetic techniques, can be used toprovide for transcription of the polynucleotides of the invention. A“promoter” typically means a minimal sequence element sufficient todirect transcription.

Nucleic acids may be inserted into a plasmid for transformation into ahost cell and for subsequent expression and/or genetic manipulation. Aplasmid is a nucleic acid that can be stably propagated in a host cell;plasmids may optionally contain expression control elements in order todrive expression of the nucleic acid. For purposes of this invention, avector is synonymous with a plasmid. Plasmids and vectors generallycontain at least an origin of replication for propagation in a cell anda promoter. Plasmids and vectors may also include an expression controlelement for expression in a host cell, and are therefore useful forexpression and/or genetic manipulation of nucleic acids encoding peptidesequences, expressing peptide sequences in host cells and organisms(e.g., a subject in need of treatment), or producing peptide sequences,for example.

As used herein, the term “transgene” means a polynucleotide that hasbeen introduced into a cell or organism by artifice. For example, a cellhaving a transgene, the transgene has been introduced by geneticmanipulation or “transformation” of the cell. A cell or progeny thereofinto which the transgene has been introduced is referred to as a“transformed cell” or “transformant.” Typically, the transgene isincluded in progeny of the transformant or becomes a part of theorganism that develops from the cell. Transgenes may be inserted intothe chromosomal DNA or maintained as a self-replicating plasmid, YAC,minichromosome, or the like.

Bacterial system promoters include T7 and inducible promoters such as pLof bacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter) andtetracycline responsive promoters. Insect cell system promoters includeconstitutive or inducible promoters (e.g., ecdysone). Mammalian cellconstitutive promoters include SV40, RSV, bovine papilloma virus (BPV)and other virus promoters, or inducible promoters derived from thegenome of mammalian cells (e.g., metallothionein IIA promoter; heatshock promoter) or from mammalian viruses (e.g., the adenovirus latepromoter; the inducible mouse mammary tumor virus long terminal repeat).Alternatively, a retroviral genome can be genetically modified forintroducing and directing expression of a peptide sequence inappropriate host cells.

As methods and uses of the invention include in vivo delivery,expression systems further include vectors designed for in vivo use.Particular non-limiting examples include adenoviral vectors (U.S. Pat.Nos. 5,700,470 and 5,731,172), adeno-associated vectors (U.S. Pat. No.5,604,090), herpes simplex virus vectors (U.S. Pat. No. 5,501,979),retroviral vectors (U.S. Pat. Nos. 5,624,820, 5,693,508 and 5,674,703),BPV vectors (U.S. Pat. No. 5,719,054), CMV vectors (U.S. Pat. No.5,561,063) and parvovirus, rotavirus, Norwalk virus and lentiviralvectors (see, e.g., U.S. Pat. No. 6,013,516). Vectors include those thatdeliver genes to cells of the intestinal tract, including the stem cells(Croyle et al., Gene Ther. 5:645 (1998); S. J. Henning, Adv. Drug Deliv.Rev. 17:341 (1997), U.S. Pat. Nos. 5,821,235 and 6,110,456). Many ofthese vectors have been approved for human studies.

Yeast vectors include constitutive and inducible promoters (see, e.g.,Ausubel et al., In: Current Protocols in Molecular Biology, Vol. 2, Ch.13, ed., Greene Publish. Assoc. & Wiley Interscience, 1988; Grant et al.Methods in Enzymology, 153:516 (1987), eds. Wu & Grossman; BitterMethods in Enzymology, 152:673 (1987), eds. Berger & Kimmel, Acad.Press, N.Y.; and, Strathern et al., The Molecular Biology of the YeastSaccharomyces (1982) eds. Cold Spring Harbor Press, Vols. I and II). Aconstitutive yeast promoter such as ADH or LEU2 or an inducible promotersuch as GAL may be used (R. Rothstein In: DNA Cloning, A PracticalApproach, Vol. 11, Ch. 3, ed. D. M. Glover, IRL Press, Wash., D.C.,1986). Vectors that facilitate integration of foreign nucleic acidsequences into a yeast chromosome, via homologous recombination forexample, are known in the art. Yeast artificial chromosomes (YAC) aretypically used when the inserted polynucleotides are too large for moreconventional vectors (e.g., greater than about 12 Kb).

Expression vectors also can contain a selectable marker conferringresistance to a selective pressure or identifiable marker (e.g.,beta-galactosidase), thereby allowing cells having the vector to beselected for, grown and expanded. Alternatively, a selectable marker canbe on a second vector that is co-transfected into a host cell with afirst vector containing a nucleic acid encoding a peptide sequence.Selection systems include but are not limited to herpes simplex virusthymidine kinase gene (Wigler et al., Cell 11:223 (1977)),hypoxanthine-guanine phosphoribosyltransferase gene (Szybalska et al.,Proc. Natl. Acad. Sci. USA 48:2026 (1962)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes thatcan be employed in tk-, hgprt- or aprt-cells, respectively.Additionally, antimetabolite resistance can be used as the basis ofselection for dhfr, which confers resistance to methotrexate (O'Hare etal., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); the gpt gene, whichconfers resistance to mycophenolic acid (Mulligan et al., Proc. Natl.Acad. Sci. USA 78:2072 (1981)); neomycin gene, which confers resistanceto aminoglycoside G-418 (Colberre-Garapin et al., J. Mol. Biol.150:1(1981)); puromycin; and hygromycin gene, which confers resistanceto hygromycin (Santerre et al., Gene 30:147 (1984)). Additionalselectable genes include trpB, which allows cells to utilize indole inplace of tryptophan; hisD, which allows cells to utilize histinol inplace of histidine (Hartman et al., Proc. Natl. Acad. Sci. USA 85:8047(1988)); and ODC (ornithine decarboxylase), which confers resistance tothe ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine,DFMO (McConlogue (1987) In: Current Communications in Molecular Biology,Cold Spring Harbor Laboratory).

In accordance with the invention, there are provided transformed cell(s)(in vitro, ex vivo and in vivo) and host cells that produce a variant orfusion of FGF19 and/or FGF21 as set forth herein, where expression ofthe variant or fusion of FGF19 and/or FGF21 is conferred by a nucleicacid encoding the variant or fusion of FGF19 and/or FGF21. Transformedand host cells that express invention peptide sequences typicallyinclude a nucleic acid that encodes the invention peptide sequence. Inone embodiment, a transformed or host cell is a prokaryotic cell. Inanother embodiment, a transformed or host cell is a eukaryotic cell. Invarious aspects, the eukaryotic cell is a yeast or mammalian (e.g.,human, primate, etc.) cell.

As used herein, a “transformed” or “host” cell is a cell into which anucleic acid is introduced that can be propagated and/or transcribed forexpression of an encoded peptide sequence. The term also includes anyprogeny or subclones of the host cell.

Transformed and host cells include but are not limited to microorganismssuch as bacteria and yeast; and plant, insect and mammalian cells. Forexample, bacteria transformed with recombinant bacteriophage nucleicacid, plasmid nucleic acid or cosmid nucleic acid expression vectors;yeast transformed with recombinant yeast expression vectors; plant cellsystems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid); insect cell systems infected with recombinant virus expressionvectors (e.g., baculovirus); and animal cell systems infected withrecombinant virus expression vectors (e.g., retroviruses, adenovirus,vaccinia virus), or transformed animal cell systems engineered fortransient or stable propagation or expression.

For gene therapy uses and methods, a transformed cell can be in asubject. A cell in a subject can be transformed with a nucleic acid thatencodes an invention peptide sequence as set forth herein in vivo.Alternatively, a cell can be transformed in vitro with a transgene orpolynucleotide, and then transplanted into a tissue of subject in orderto effect treatment. Alternatively, a primary cell isolate or anestablished cell line can be transformed with a transgene orpolynucleotide that encodes a variant of FGF19 and/or FGF21 or afusion/chimeric sequence (or variant) thereof, such as a chimericpeptide sequence including all or a portion of FGF19, or including allor a portion of FGF21, and then optionally transplanted into a tissue ofa subject.

Non-limiting target cells for expression of peptide sequences,particularly for expression in vivo, include pancreas cells (isletcells), muscle cells, mucosal cells and endocrine cells. Such endocrinecells can provide inducible production (secretion) of a variant of FGF19and/or FGF21, or a fusion/chimeric sequence (or variant) thereof, suchas a chimeric peptide sequence including all or a portion of FGF19, orincluding all or a portion of FGF21. Additional cells to transforminclude stem cells or other multipotent or pluripotent cells, forexample, progenitor cells that differentiate into the various pancreascells (islet cells), muscle cells, mucosal cells and endocrine cells.Targeting stem cells provides longer term expression of peptidesequences of the invention.

As used herein, the term “cultured,” when used in reference to a cell,means that the cell is grown in vitro. A particular example of such acell is a cell isolated from a subject, and grown or adapted for growthin tissue culture. Another example is a cell genetically manipulated invitro, and transplanted back into the same or a different subject.

The term “isolated,” when used in reference to a cell, means a cell thatis separated from its naturally occurring in vivo environment.“Cultured” and “isolated” cells may be manipulated by the hand of man,such as genetically transformed. These terms include any progeny of thecells, including progeny cells that may not be identical to the parentalcell due to mutations that occur during cell division. The terms do notinclude an entire human being.

Nucleic acids encoding invention peptide sequences can be introduced forstable expression into cells of a whole organism. Such organismsincluding non-human transgenic animals are useful for studying theeffect of peptide expression in a whole animal and therapeutic benefit.For example, as disclosed herein, production of a variant of FGF19and/or FGF21 or a fusion/chimeric sequence (or variant) thereof, such asa chimeric peptide sequence including all or a portion of FGF19, orincluding all or a portion of FGF21 as set forth herein, in micemodulated bile acid homeostasis.

Mice strains that develop or are susceptible to developing a particulardisease (e.g., diabetes, degenerative disorders, cancer, etc.) are alsouseful for introducing therapeutic proteins as described herein in orderto study the effect of therapeutic protein expression in the diseasesusceptible mouse. Transgenic and genetic animal models that aresusceptible to particular disease or physiological conditions, such asstreptozotocin (STZ)-induced diabetic (STZ) mice, are appropriatetargets for expressing variants of FGF19 and/or FGF21, fusions/chimericsequences (or variant) thereof, such as a chimeric peptide sequenceincluding all or a portion of FGF19, or including all or a portion ofFGF21, as set forth herein. Thus, in accordance with the invention,there are provided non-human transgenic animals that produce a variantof FGF19 and/or FGF21, or a fusion/chimeric sequence (or variant)thereof, such as a chimeric peptide sequence including all or a portionof FGF19, or including all or a portion of FGF21, the production ofwhich is not naturally occurring in the animal which is conferred by atransgene present in somatic or germ cells of the animal.

The term “transgenic animal” refers to an animal whose somatic or germline cells bear genetic information received, directly or indirectly, bydeliberate genetic manipulation at the subcellular level, such as bymicroinjection or infection with recombinant virus. The term“transgenic” further includes cells or tissues (i.e., “transgenic cell,”“transgenic tissue”) obtained from a transgenic animal geneticallymanipulated as described herein. In the present context, a “transgenicanimal” does not encompass animals produced by classical crossbreedingor in vitro fertilization, but rather denotes animals in which one ormore cells receive a nucleic acid molecule. Invention transgenic animalscan be either heterozygous or homozygous with respect to the transgene.Methods for producing transgenic animals, including mice, sheep, pigsand frogs, are well known in the art (see, e.g., U.S. Pat. Nos.5,721,367, 5,695,977, 5,650,298, and 5,614,396) and, as such, areadditionally included.

Peptide sequences, nucleic acids encoding peptide sequences, vectors andtransformed host cells expressing peptide sequences include isolated andpurified forms. The term “isolated,” when used as a modifier of aninvention composition, means that the composition is separated,substantially completely or at least in part, from one or morecomponents in an environment. Generally, compositions that exist innature, when isolated, are substantially free of one or more materialswith which they normally associate with in nature, for example, one ormore protein, nucleic acid, lipid, carbohydrate or cell membrane. Theterm “isolated” does not exclude alternative physical forms of thecomposition, such as variants, modifications or derivatized forms,fusions and chimeras, multimers/oligomers, etc., or forms expressed inhost cells. The term “isolated” also does not exclude forms (e.g.,pharmaceutical compositions, combination compositions, etc.) in whichthere are combinations therein, any one of which is produced by the handof man.

An “isolated” composition can also be “purified” when free of some, asubstantial number of, or most or all of one or more other materials,such as a contaminant or an undesired substance or material. Peptidesequences of the invention are generally not known or believed to existin nature. However, for a composition that does exist in nature, anisolated composition will generally be free of some, a substantialnumber of, or most or all other materials with which it typicallyassociates with in nature. Thus, an isolated peptide sequence that alsooccurs in nature does not include polypeptides or polynucleotidespresent among millions of other sequences, such as proteins of a proteinlibrary or nucleic acids in a genomic or cDNA library, for example. A“purified” composition includes combinations with one or more otherinactive or active molecules. For example, a peptide sequence of theinvention combined with another drug or agent, such as a glucoselowering drug or therapeutic agent, for example.

As used herein, the term “recombinant,” when used as a modifier ofpeptide sequences, nucleic acids encoding peptide sequences, etc., meansthat the compositions have been manipulated (i.e., engineered) in afashion that generally does not occur in nature (e.g., in vitro). Aparticular example of a recombinant peptide would be where a peptidesequence of the invention is expressed by a cell transfected with anucleic acid encoding the peptide sequence. A particular example of arecombinant nucleic acid would be where a nucleic acid (e.g., genomic orcDNA) encoding a peptide sequence cloned into a plasmid, with or without5′,3′ or intron regions that the gene is normally contiguous within thegenome of the organism. Another example of a recombinant peptide ornucleic acid is a hybrid or fusion sequence, such as a chimeric peptidesequence comprising a portion of FGF19 and a portion of FGF21.

In accordance with the invention, there are provided compositions andmixtures of invention peptide sequences, including subsequences,variants and modified forms of the exemplified peptide sequences(including the FGF19 and FGF21 variants and subsequences listed inTables 1-10 and the Sequence Listing, and the FGF19/FGF21 fusions andchimeras listed in Tables 1-10 and the Sequence Listing). In oneembodiment, a mixture includes one or more peptide sequences and apharmaceutically acceptable carrier or excipient. In another embodiment,a mixture includes one or more peptide sequences and an adjunct drug ortherapeutic agent, such as a bile acid homeostasis modulating oranti-diabetic, or glucose lowering, drug or therapeutic agent.Combinations, such as one or more peptide sequences in apharmaceutically acceptable carrier or excipient, with one or more of abile acid homeostasis modulating or a treatment for a bile-acid relatedor associated disorder, or anti-diabetic, or glucose lowering drug ortherapeutic agent are also provided. Such combinations of peptidesequence of the invention with another drug or agent, such as a bileacid homeostasis modulating or acid related or associated disordertreating, or glucose lowering drug or therapeutic agent, for example areuseful in accordance with the invention methods and uses, for example,for treatment of a subject.

Combinations also include incorporation of peptide sequences or nucleicacids of the invention into particles or a polymeric substances, such aspolyesters, carbohydrates, polyamine acids, hydrogel, polyvinylpyrrolidone, ethylene-vinylacetate, methylcellulose,carboxymethylcellulose, protamine sulfate, or lactide/glycolidecopolymers, polylactide/glycolide copolymers, or ethylenevinylacetatecopolymers; entrapment in microcapsules prepared by coacervationtechniques or by interfacial polymerization, for example, by the use ofhydroxymethylcellulose or gelatin-microcapsules, or poly(methylmethacrolate) microcapsules, respectively; incorporation incolloid drug delivery and dispersion systems such as macromoleculecomplexes, nano-capsules, microspheres, beads, and lipid-based systems(e.g., N-fatty acyl groups such as N-lauroyl, N-oleoyl, fatty aminessuch as dodecyl amine, oleoyl amine, etc., see U.S. Pat. No. 6,638,513),including oil-in-water emulsions, micelles, mixed micelles, andliposomes, for example.

Invention peptides including subsequences, variants and modified formsof the exemplified peptide sequences (including the FGF19 and FGF21variants and subsequences listed in Tables 1-10 and the SequenceListing, and the FGF19/FGF21 fusions and chimeras listed in Tables 1-10and the Sequence Listing) as set forth herein can be used to modulateglucose metabolism and facilitate transport of glucose from the blood tokey metabolic organs such as muscle, liver and fat. Such peptidesequences can be produced in amounts sufficient or effective to restoreglucose tolerance and/or to improve or provide normal glucosehomeostasis.

As disclosed herein, administration of various FGF19 and/FGF21 variantsand fusion peptide sequences to mice successfully modulated bile acidhomeostasis. Furthermore, in contrast to FGF19, certain peptidesequences did not stimulate or induce HCC formation or tumorigenesis inmice. Thus, administration of invention peptides, includingsubsequences, variants and modified forms of the exemplified peptidesequences (including the FGF19 and FGF21 variants and subsequenceslisted in Tables 1-10 and the Sequence Listing, and the FGF19/FGF21fusions and chimeras listed in Tables 1-10 and the Sequence Listing),into an animal, either by direct or indirect in vivo or by ex vivomethods (e.g., administering the variant or fusion peptide, a nucleicacid encoding the variant or fusion peptide, or a transformed cell orgene therapy vector expressing the variant or fusion peptide), can beused to treat various disorders, such as bile-acid related or associateddisorders.

Accordingly, the invention includes in vitro, ex vivo and in vivo (e.g.,on or in a subject) methods and uses. Such methods and uses can bepracticed with any of the peptide sequences of the invention set forthherein.

In accordance with the invention, there are provided methods of treatinga subject having, or at risk of having, a disorder. In variousembodiments, a method includes administering a peptide sequence, such asan FGF19 or FGF21 variant, fusion or chimera disclosed herein (see,e.g., Tables 1-10), or a subsequence, a variant or modified form of anFGF19 or FGF21 variant, fusion or chimera disclosed herein (see, e.g.,Tables 1-10 and the Sequence Listing), to a subject in an amounteffective for treating the disorder.

Exemplary disorders treatable, preventable, and the like with inventionpeptides, and methods and uses, include bile-acid related or associateddisorders. Non limiting examples of diseases and disorders include:metabolic syndrome; a lipid- or glucose-related disorder; cholesterol ortriglyceride metabolism; type 2 diabetes; cholestasis, including, forexample diseases of intrahepatic cholestasis (e.g., PBC, PFIC, PSC, PIC,neonatal cholestasis, and drug induced cholestasis (e.g., estrogen)),and diseases of extrahepatic cholestasis (e.g., bile cut compressionfrom tumor, bile duct blockade by gall stones); bile acid malabsorptionand other disorders involving the distal small intestine, includingileal resection, inflammatory bowel diseases (e.g., Crohn's disease andulcerative colitis), disorders impairing absorption of bile acids nototherwise characterized (idiopathic)) leading to diarrhea (e.g., BAD)and GI symptoms, and GI, liver, and/or biliary cancers (e.g., coloncancer and hepatocellular cancer); and/or bile acid synthesisabnormalities, such as those contributing to NASH, cirrhosis and portalhypertension. For treatment, invention peptide sequences can beadministered to subjects in need of modulation of bile acid homeostasisor having a bile-acid related or associated disorder. Peptide sequencesof the invention may also be useful in other hyperglycemic-relateddisorders, including kidney damage (e.g., tubule damage or nephropathy),liver degeneration, eye damage (e.g., diabetic retinopathy orcataracts), and diabetic foot disorders; Dyslipidemias and theirsequelae such as, for example, atherosclerosis, coronary artery disease,cerebrovascular disorders and the like.

Other conditions which may be associated with metabolic syndrome, suchas obesity and elevated body mass (including the co-morbid conditionsthereof such as, but not limited to, nonalcoholic fatty liver disease(NAFLD), nonalcoholic steatohepatitis (NASH), and polycystic ovariansyndrome (PCOS)), and also include thromboses, hypercoagulable andprothrombotic states (arterial and venous), hypertension (includingportal hypertension (defined as a hepatic venous pressure gradient(HVPG) greater than 5 mm Hg), cardiovascular disease, stroke and heartfailure; Disorders or conditions in which inflammatory reactions areinvolved, including atherosclerosis, chronic inflammatory bowel diseases(e.g., Crohn's disease and ulcerative colitis), asthma, lupuserythematosus, arthritis, or other inflammatory rheumatic disorders;Disorders of cell cycle or cell differentiation processes such asadipose cell tumors, lipomatous carcinomas including, for example,liposarcomas, solid tumors, and neoplasms; Neurodegenerative diseasesand/or demyelinating disorders of the central and peripheral nervoussystems and/or neurological diseases involving neuroinflammatoryprocesses and/or other peripheral neuropathies, including Alzheimer'sdisease, multiple sclerosis, Parkinson's disease, progressive multifocalleukoencephalopathy and Guillian-Barre syndrome; Skin and dermatologicaldisorders and/or disorders of wound healing processes, includingerythemato-squamous dermatoses; and other disorders such as syndrome X,osteoarthritis, and acute respiratory distress syndrome.

As used herein, the term “bile-acid related or associated disorder,”when used in reference to a condition of a subject means a transient orchronic abnormal level of a bile acid (one or more bile acids) presentin the subject. The condition can be caused by inhibition, reduction ora delay in bile acid synthesis, metabolism or absorption such that thesubject exhibits a bile acid level not typically found in normalsubjects.

As disclosed herein, the invention includes methods of preventing (e.g.,in subjects predisposed to having a particular disorder(s)), delaying,slowing or inhibiting progression of, the onset of, or treating (e.g.,ameliorating) a bile-acid related or associated disorder relative to anappropriate matched subject of comparable age, gender, race, etc.).Thus, in various embodiments, a method of the invention for, forexample, modulating bile acid homeostasis or treating a bile-acidrelated or associated disorder includes contacting or administering apeptide of the invention as set forth herein (e.g., a variant or fusionof FGF19 and/or FGF21 as set forth in Tables 1-10 or the SequenceListing, for example) in an amount effective to modulate bile acidhomeostasis or treat a bile-acid related or associated disorder.

Moreover, the invention includes methods of preventing (e.g., insubjects predisposed to having a particular disorder(s)), slowing orinhibiting the progression of, delaying the onset of, or treatingundesirable levels or abnormally low levels of bile acids, all of which,alone or in combination, can lead to, for example, to at a bile-acidrelated or associated disorder. Such disorders can be due to, forexample, genetic predisposition or diet, for example.

The term “subject” refers to an animal. Typically, the animal is amammal that would benefit from treatment with a peptide sequence of theinvention. Particular examples include primates (e.g., humans), dogs,cats, horses, cows, pigs, and sheep.

Subjects include those having a disorder, e.g., a bile acid related orassociated disorder, such as metabolic syndrome; a lipid- orglucose-related disorder; cholesterol or triglyceride metabolism; type 2diabetes; cholestasis, including, for example diseases of intrahepaticcholestasis (e.g., PBC, PFIC, PSC, PIC, neonatal cholestasis, and druginduced cholestasis (e.g., estrogen)), and diseases of extrahepaticcholestasis (e.g., bile cut compression from tumor, bile duct blockadeby gall stones); bile acid malabsorption and other disorders involvingthe distal small intestine, including ileal resection, inflammatorybowel diseases (e.g., Crohn's disease and ulcerative colitis), disordersimpairing absorption of bile acids not otherwise characterized(idiopathic)) leading to diarrhea (e.g., BAD) and GI symptoms, and GI,liver, and/or biliary cancers (e.g., colon cancer and hepatocellularcancer); and/or bile acid synthesis abnormalities, such as thosecontributing to NASH, cirrhosis and portal hypertension; or subjectsthat do not have a disorder but may be at risk of developing thedisorder. Subjects at risk of developing a bile acid associated orrelated disorder include, for example, those whose diet may contributeto development of acute or metabolic syndrome; a lipid- orglucose-related disorder; cholesterol or triglyceride metabolism; type 2diabetes; cholestasis, including, for example diseases of intrahepaticcholestasis (e.g., PBC, PFIC, PSC, PIC, neonatal cholestasis, and druginduced cholestasis (e.g., estrogen)), and diseases of extrahepaticcholestasis (e.g., bile cut compression from tumor, bile duct blockadeby gall stones); bile acid malabsorption and other disorders involvingthe distal small intestine, including ileal resection, inflammatorybowel diseases (e.g., Crohn's disease and ulcerative colitis), disordersimpairing absorption of bile acids not otherwise characterized(idiopathic)) leading to diarrhea (e.g., BAD) and GI symptoms, and GI,liver, and/or biliary cancers (e.g., colon cancer and hepatocellularcancer); and/or bile acid synthesis abnormalities, such as thosecontributing to NASH, cirrhosis and portal hypertension; as well asthose which may have a family history or genetic predisposition towardsdevelopment of a bile acid related or associated disorder, such asmetabolic syndrome; a lipid- or glucose-related disorder; cholesterol ortriglyceride metabolism; type 2 diabetes; cholestasis, including, forexample diseases of intrahepatic cholestasis (e.g., PBC, PFIC, PSC, PIC,neonatal cholestasis, and drug induced cholestasis (e.g., estrogen)),and diseases of extrahepatic cholestasis (e.g., bile cut compressionfrom tumor, bile duct blockade by gall stones); bile acid malabsorptionand other disorders involving the distal small intestine, includingileal resection, inflammatory bowel diseases (e.g., Crohn's disease andulcerative colitis), disorders impairing absorption of bile acids nototherwise characterized (idiopathic)) leading to diarrhea (e.g., BAD)and GI symptoms, and GI, liver, and/or biliary cancers (e.g., coloncancer and hepatocellular cancer); and/or bile acid synthesisabnormalities, such as those contributing to NASH, cirrhosis and portalhypertension.

As disclosed herein, treatment methods include contacting oradministering a peptide of the invention as set forth herein (e.g., avariant or fusion of FGF19 and or FGF21 as set forth in Tables 1-10 orthe Sequence Listing, for example) in an amount effective to achieve adesired outcome or result in a subject. A treatment that results in adesired outcome or result includes decreasing, reducing or preventingseverity or frequency of one or more symptoms of the condition in thesubject, e.g., an improvement in the subject's condition or a“beneficial effect” or “therapeutic effect.” Therefore, treatment candecrease or reduce or prevent the severity or frequency of one or moresymptoms of the disorder, stabilize or inhibit progression or worseningof the disorder, and in some instances, reverse the disorder,transiently (e.g., for 1-6, 6-12, or 12-24 hours), for medium term(e.g., 1-6, 6-12, 12-24 or 24-48 days) or long term (e.g., for 1-6,6-12, 12-24, 24-48 weeks, or greater than 24-48 weeks). Thus, in thecase of a bile acid related or associated disorder, such as metabolicsyndrome; a lipid- or glucose-related disorder; cholesterol ortriglyceride metabolism; type 2 diabetes; cholestasis, including, forexample diseases of intrahepatic cholestasis (e.g., PBC, PFIC, PSC, PIC,neonatal cholestasis, and drug induced cholestasis (e.g., estrogen)),and diseases of extrahepatic cholestasis (e.g., bile cut compressionfrom tumor, bile duct blockade by gall stones); bile acid malabsorptionand other disorders involving the distal small intestine, includingileal resection, inflammatory bowel diseases (e.g., Crohn's disease andulcerative colitis), disorders impairing absorption of bile acids nototherwise characterized (idiopathic)) leading to diarrhea (e.g., BAD)and GI symptoms, and GI, liver, and/or biliary cancers (e.g., coloncancer and hepatocellular cancer); and/or bile acid synthesisabnormalities, such as those contributing to NASH, cirrhosis and portalhypertension; for example, treatment can lower or reduce one or moresymptoms or effects of the bile acid associated or related disorder.

An “effective amount” or a “sufficient amount” for use and/or fortreating a subject refer to an amount that provides, in single ormultiple doses, alone, or in combination with one or more othercompositions (therapeutic agents such as a drug or treatment forhyperglycemia), treatments, protocols, or therapeutic regimens agents, adetectable response of any duration of time (transient, medium or longterm), a desired outcome in or an objective or subjective benefit to asubject of any measurable or detectable degree or for any duration oftime (e.g., for hours, days, months, years, or cured). Such amountstypically are effective to ameliorate a disorder, or one, multiple orall adverse symptoms, consequences or complications of the disorder, toa measurable extent, although reducing or inhibiting a progression orworsening of the disorder, is considered a satisfactory outcome.

As used herein, the term “ameliorate” means an improvement in thesubject's disorder, a reduction in the severity of the disorder, or aninhibition of progression or worsening of the disorder (e.g.,stabilizing the disorder). In the case of a bile acid related orassociated disorder (e.g., metabolic syndrome; a lipid- orglucose-related disorder; cholesterol or triglyceride metabolism; type 2diabetes; cholestasis, including, for example diseases of intrahepaticcholestasis (e.g., PBC, PFIC, PSC, PIC, neonatal cholestasis, and druginduced cholestasis (e.g., estrogen)), and diseases of extrahepaticcholestasis (e.g., bile cut compression from tumor, bile duct blockadeby gall stones); bile acid malabsorption and other disorders involvingthe distal small intestine, including ileal resection, inflammatorybowel diseases (e.g., Crohn's disease and ulcerative colitis), disordersimpairing absorption of bile acids not otherwise characterized(idiopathic)) leading to diarrhea (e.g., BAD) and GI symptoms, and GI,liver, and/or biliary cancers (e.g., colon cancer and hepatocellularcancer); and/or bile acid synthesis abnormalities, such as thosecontributing to NASH, cirrhosis and portal hypertension; for example, animprovement can be a lowering or a reduction in one or more symptoms oreffects of the disorder.

A therapeutic benefit or improvement therefore need not be completeablation of any one, most or all symptoms, complications, consequencesor underlying causes associated with the disorder or disease. Thus, asatisfactory endpoint is achieved when there is a transient, medium orlong term, incremental improvement in a subject's condition, or apartial reduction in the occurrence, frequency, severity, progression,or duration, or inhibition or reversal, of one or more associatedadverse symptoms or complications or consequences or underlying causes,worsening or progression (e.g., stabilizing one or more symptoms orcomplications of the condition, disorder or disease), of the disorder ordisease, over a duration of time (hours, days, weeks, months, etc.).

Thus, in the case of a disorder treatable by a peptide sequence of theinvention, the amount of peptide sufficient to ameliorate a disorderwill depend on the type, severity and extent, or duration of thedisorder, the therapeutic effect or outcome desired, and can be readilyascertained by the skilled artisan. Appropriate amounts will also dependupon the individual subject (e.g., the bioavailability within thesubject, gender, age, etc.). For example, a transient, or partial,restoration of normal bile acid homeostasis in a subject can reduce thedosage amount or frequency of a drug used to treat metabolic syndrome; alipid- or glucose-related disorder; cholesterol or triglyceridemetabolism; type 2 diabetes; cholestasis, including, for examplediseases of intrahepatic cholestasis (e.g., PBC, PFIC, PSC, PIC,neonatal cholestasis, and drug induced cholestasis (e.g., estrogen)),and diseases of extrahepatic cholestasis (e.g., bile cut compressionfrom tumor, bile duct blockade by gall stones); bile acid malabsorptionand other disorders involving the distal small intestine, includingileal resection, inflammatory bowel diseases (e.g., Crohn's disease andulcerative colitis), disorders impairing absorption of bile acids nototherwise characterized (idiopathic)) leading to diarrhea (e.g., BAD)and GI symptoms, and GI, liver, and/or biliary cancers (e.g., coloncancer and hepatocellular cancer); and/or bile acid synthesisabnormalities, such as those contributing to NASH, cirrhosis and portalhypertension; even though complete freedom from treatment has notresulted. An effective amount can be ascertained, for example, bymeasuring one or more relevant physiological effects.

Methods and uses of the invention for treating a subject are applicablefor prophylaxis to prevent or reduce likelihood of a disorder in asubject, such as a bile acid related or associated disorder.Alternatively, methods and uses can be practiced during or followingtreatment of a subject. For example, prior to, during or followingtreatment of a subject to improve bile acid homeostasis using anotherdrug or therapeutic agent, for example, a method or use of the inventioncan, for example, a peptide sequence of the invention can beadministered to the subject. In addition, a composition such as apeptide sequence of the invention can be combined with another drug oragent, such as a bile acid stabilizing drug or therapeutic agent, forexample.

Accordingly, methods and uses of the invention for treating a subjectcan be practiced prior to, substantially contemporaneously with orfollowing another treatment, and can be supplemented with other forms oftherapy. Supplementary therapies include other glucose loweringtreatments, such as insulin, an insulin sensitivity enhancer and otherdrug treatments, a change in diet (low sugar, fats, etc.), weight losssurgery—(reducing stomach volume by gastric bypass, gastrectomy),gastric banding, gastric balloon, gastric sleeve, etc. For example, amethod or use of the invention for treating a hyperglycemic or insulinresistance disorder can be used in combination with drugs or otherpharmaceutical compositions that lower glucose or increase insulinsensitivity in a subject.

The present disclosure contemplates combination therapy with numerousagents (and classes thereof), including 1) insulin e.g., bolus and basalanalogs), insulin mimetics and agents that entail stimulation of insulinsecretion, including sulfonylureas (e.g., chlorpropamide, tolazamide,acetohexamide, tolbutamide, glyburide, glimepiride, glipizide) andmeglitinides (e.g., repaglinide (PRANDIN) and nateglinide (STARLIX)); 2)biguanides (e.g., metformin (GLUCOPHAGE)) and other agents that act bypromoting glucose utilization, reducing hepatic glucose productionand/or diminishing intestinal glucose output; 3) alpha-glucosidaseinhibitors (e.g., acarbose and miglitol) and other agents that slow downcarbohydrate digestion and consequently absorption from the gut andreduce postprandial hyperglycemia; 4) thiazolidinediones (e.g.,rosiglitazone (AVANDIA), troglitazone (REZULIN), pioglitazone (ACTOS),glipizide, balaglitazone, rivoglitazone, netoglitazone, troglitazone,englitazone, ciglitazone, adaglitazone, darglitazone that enhanceinsulin action (e.g., by insulin sensitization), thus promoting glucoseutilization in peripheral tissues; 5) glucagon-like-peptides includingDPP-IV inhibitors (e.g., vildagliptin (GALVUS) and sitagliptin(JANUVIA)) and Glucagon-Like Peptide-1 (GLP-1) and GLP-1 agonists andanalogs (e.g., exenatide (BYETTA and ITCA 650 (an osmotic pump insertedsubcutaneously that delivers an exenatide analog over a 12-month period;Intarcia, Boston, Mass.)); 6) and DPP-IV-resistant analogues (incretinmimetics), PPAR gamma agonists, dual-acting PPAR agonists, pan-actingPPAR agonists, PTP1B inhibitors, SGLT inhibitors, insulin secretagogues,RXR agonists, glycogen synthase kinase-3 inhibitors, immune modulators,beta-3 adrenergic receptor agonists, 11beta-HSD1 inhibitors, and amylinanalogues.

Other exemplary agents that can be used, in certain embodiments, incombination with the chimeric peptides and methods provided hereininclude dipeptidyl peptidase-4 (DPP-4) inhibitors, bromocriptineformulations (e.g. and bile acid sequestrants (e.g., colesevelam), andSGLT-2 inhibitors. Appetite suppression drugs are also well known andcan be used in combination with the compositions and methods providedherein. Supplementary therapies can be administered prior to,contemporaneously with or following invention methods and uses.

Peptide sequences of the invention including subsequences, sequencevariants and modified forms of the exemplified peptide sequences(sequences listed in Tables 1-10 and the Sequence Listing), may beformulated in a unit dose or unit dosage form. In a particularembodiment, a peptide sequence is in an amount effective to treat asubject in need of treatment, e.g., due to abnormal or aberrant bileacid homeostasis, such as metabolic syndrome; a lipid- orglucose-related disorder; cholesterol or triglyceride metabolism; type 2diabetes; cholestasis, including, for example diseases of intrahepaticcholestasis (e.g., PBC, PFIC, PSC, PIC, neonatal cholestasis, and druginduced cholestasis (e.g., estrogen)), and diseases of extrahepaticcholestasis (e.g., bile cut compression from tumor, bile duct blockadeby gall stones); bile acid malabsorption and other disorders involvingthe distal small intestine, including ileal resection, inflammatorybowel diseases (e.g., Crohn's disease and ulcerative colitis), disordersimpairing absorption of bile acids not otherwise characterized(idiopathic)) leading to diarrhea (e.g., BAD) and GI symptoms, and GI,liver, and/or biliary cancers (e.g., colon cancer and hepatocellularcancer); and/or bile acid synthesis abnormalities, such as thosecontributing to NASH, cirrhosis and portal hypertension. Exemplary unitdoses range from about 25-250, 250-500, 500-1000, 1000-2500 or2500-5000, 5000-25,000, 25,000-50,000 ng; from about 25-250, 250-500,500-1000, 1000-2500 or 2500-5000, 5000-25,000, 25,000-50,000 μg; andfrom about 25-250, 250-500, 500-1000, 1000-2500 or 2500-5000,5000-25,000, 25,000-50,000 mg.

Peptide sequences of the invention including subsequences, sequencevariants and modified forms of the exemplified peptide sequences(sequences listed in Tables 1-10 and the Sequence Listing) can beadministered to provide the intended effect as a single dose or multipledosages, for example, in an effective or sufficient amount. Exemplarydoses range from about 25-250, 250-500, 500-1000, 1000-2500 or2500-5000, 5000-25,000, 25,000-50,000 pg/kg; from about 50-500,500-5000, 5000-25,000 or 25,000-50,000 ng/kg; and from about 25-250,250-500, 500-1000, 1000-2500 or 2500-5000, 5000-25,000, 25,000-50,000μg/kg. Single or multiple doses can be administered, for example,multiple times per day, on consecutive days, alternating days, weekly orintermittently (e.g., twice per week, once every 1, 2, 3, 4, 5, 6, 7 or8 weeks, or once every 2, 3, 4, 5 or 6 months).

Peptide sequences of the invention including subsequences, variants andmodified forms of the exemplified peptide sequences (sequences listed inTables 1-10 and the Sequence Listing) can be administered and methodsmay be practiced via systemic, regional or local administration, by anyroute. For example, a peptide sequence can be administered parenterally(e.g., subcutaneously, intravenously, intramuscularly, orintraperitoneally), orally (e.g., ingestion, buccal, or sublingual),inhalation, intradermally, intracavity, intracranially, transdermally(topical), transmucosally or rectally. Peptide sequences of theinvention including subsequences, variants and modified forms of theexemplified peptide sequences (sequences listed in Tables 1-10 and theSequence Listing) and methods of the invention including pharmaceuticalcompositions can be administered via a (micro)encapsulated deliverysystem or packaged into an implant for administration.

A particular non-limiting example of parenteral (e.g., subcutaneous)administration entails the use of Intarcia's subcutaneous deliverysystem (Intarcia Therapeutics, Inc.; Hayward, Calif.). The systemcomprises a miniature osmotic pump that delivers a consistent amount ofa therapeutic agent over a desired period of time. In addition tomaintaining drug levels within an appropriate therapeutic range, thesystem can be used with formulations that maintain the stability ofproteinaceous therapeutic agents at human body temperature for extendedperiods of time.

The invention further provides “pharmaceutical compositions,” whichinclude a peptide sequence (or sequences) of the invention, includingsubsequences, variants and modified forms of the exemplified peptidesequences (sequences listed in Tables 1-10 and the Sequence Listing),and one or more pharmaceutically acceptable or physiologicallyacceptable diluent, carrier or excipient. In particular embodiments, apeptide sequence or sequences are present in a therapeuticallyacceptable amount. The pharmaceutical compositions may be used inaccordance with the invention methods and uses. Thus, for example, thepharmaceutical compositions can be administered ex vivo or in vivo to asubject in order to practice treatment methods and uses of theinvention.

Pharmaceutical compositions of the invention can be formulated to becompatible with the intended method or route of administration;exemplary routes of administration are set forth herein. In addition,the pharmaceutical compositions may further comprise othertherapeutically active agents or compounds disclosed herein (e.g., bileacid stabilizing agents or drugs) or known to the skilled artisan whichcan be used in the treatment or prevention of various bile acid diseasesand disorders as set forth herein.

Pharmaceutical compositions typically comprise a therapeuticallyeffective amount of at least one of the peptide sequences of theinvention, including subsequences, variants and modified forms of theexemplified peptide sequences (sequences listed in Tables 1-10 and theSequence Listing) and one or more pharmaceutically and physiologicallyacceptable formulation agents. Suitable pharmaceutically acceptable orphysiologically acceptable diluents, carriers or excipients include, butare not limited to, antioxidants (e.g., ascorbic acid and sodiumbisulfate), preservatives (e.g., benzyl alcohol, methyl parabens, ethylor n-propyl, p-hydroxybenzoate), emulsifying agents, suspending agents,dispersing agents, solvents, fillers, bulking agents, buffers, vehicles,diluents, and/or adjuvants. For example, a suitable vehicle may bephysiological saline solution or citrate buffered saline, possiblysupplemented with other materials common in pharmaceutical compositionsfor parenteral administration. Neutral buffered saline or saline mixedwith serum albumin are further exemplary vehicles. Those skilled in theart will readily recognize a variety of buffers that could be used inthe pharmaceutical compositions and dosage forms used in the invention.Typical buffers include, but are not limited to pharmaceuticallyacceptable weak acids, weak bases, or mixtures thereof. Buffercomponents also include water soluble materials such as phosphoric acid,tartaric acids, lactic acid, succinic acid, citric acid, acetic acid,ascorbic acid, aspartic acid, glutamic acid, and salts thereof.

A primary solvent in a vehicle may be either aqueous or non-aqueous innature. In addition, the vehicle may contain other pharmaceuticallyacceptable excipients for modifying or maintaining the pH, osmolarity,viscosity, sterility or stability of the pharmaceutical composition. Incertain embodiments, the pharmaceutically acceptable vehicle is anaqueous buffer. In other embodiments, a vehicle comprises, for example,sodium chloride and/or sodium citrate.

Pharmaceutical compositions of the invention may contain still otherpharmaceutically-acceptable formulation agents for modifying ormaintaining the rate of release of an invention peptide. Suchformulation agents include those substances known to artisans skilled inpreparing sustained release formulations. For further referencepertaining to pharmaceutically and physiologically acceptableformulation agents, see, for example, Remington's PharmaceuticalSciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages1435-1712, The Merck Index, 12th Ed. (1996, Merck Publishing Group,Whitehouse, N.J.); and Pharmaceutical Principles of Solid Dosage Forms(1993, Technonic Publishing Co., Inc., Lancaster, Pa.). Additionalpharmaceutical compositions appropriate for administration are known inthe art and are applicable in the methods and compositions of theinvention.

A pharmaceutical composition may be stored in a sterile vial as asolution, suspension, gel, emulsion, solid, or dehydrated or lyophilizedpowder. Such compositions may be stored either in a ready to use form, alyophilized form requiring reconstitution prior to use, a liquid formrequiring dilution prior to use, or other acceptable form. In someembodiments, a pharmaceutical composition is provided in a single-usecontainer (e.g., a single-use vial, ampoule, syringe, or autoinjector(similar to, e.g., an EpiPen®)), whereas a multi-use container (e.g., amulti-use vial) is provided in other embodiments. Any drug deliveryapparatus may be used to deliver invention peptides, including implants(e.g., implantable pumps) and catheter systems, both of which are knownto the skilled artisan. Depot injections, which are generallyadministered subcutaneously or intramuscularly, may also be utilized torelease invention peptides over a defined period of time. Depotinjections are usually either solid- or oil-based and generally compriseat least one of the formulation components set forth herein. The skilledartisan is familiar with possible formulations and uses of depotinjections.

A pharmaceutical composition can be formulated to be compatible with itsintended route of administration. Thus, pharmaceutical compositionsinclude carriers, diluents, or excipients suitable for administration byroutes including parenteral (e.g., subcutaneous (s.c.), intravenous,intramuscular, or intraperitoneal), intradermal, oral (e.g., ingestion),inhalation, intracavity, intracranial, and transdermal (topical).

Pharmaceutical compositions may be in the form of a sterile injectableaqueous or oleagenous suspension. This suspension may be formulatedusing suitable dispersing or wetting agents and suspending agentsdisclosed herein or known to the skilled artisan. The sterile injectablepreparation may also be a sterile injectable solution or suspension in anon-toxic parenterally-acceptable diluent or solvent, for example, as asolution in 1,3-butane diol. Acceptable diluents, solvents anddispersion media that may be employed include water, Ringer's solution,isotonic sodium chloride solution, Cremophor EL™ (BASF, Parsippany,N.J.) or phosphate buffered saline (PBS), ethanol, polyol (e.g.,glycerol, propylene glycol, and liquid polyethylene glycol), andsuitable mixtures thereof. In addition, sterile, fixed oils areconventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordiglycerides. Moreover, fatty acids such as oleic acid find use in thepreparation of injectables. Prolonged absorption of particularinjectable formulations can be achieved by including an agent thatdelays absorption (e.g., aluminum monostearate or gelatin).

Pharmaceutical compositions may be in a form suitable for oral use, forexample, as tablets, capsules, troches, lozenges, aqueous or oilysuspensions, dispersible powders or granules, emulsions, hard or softcapsules, or syrups, solutions, microbeads or elixirs. Pharmaceuticalcompositions intended for oral use may be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions. Such compositions may contain one or more agents such assweetening agents, flavoring agents, coloring agents and preservingagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets containing an invention peptide may be inadmixture with non-toxic pharmaceutically acceptable excipients suitablefor the manufacture of tablets. These excipients include, for example,diluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia, and lubricating agents, for example magnesiumstearate, stearic acid or talc.

Tablets, capsules and the like suitable for oral administration may beuncoated or they may be coated by known techniques to delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a timedelay material such as glyceryl monostearate or glyceryl distearate maybe employed. They may also be coated by techniques known in the art toform osmotic therapeutic tablets for controlled release. Additionalagents include biodegradable or biocompatible particles or a polymericsubstance such as polyesters, polyamine acids, hydrogel, polyvinylpyrrolidone, polyanhydrides, polyglycolic acid, ethylene-vinylacetate,methylcellulose, carboxymethylcellulose, protamine sulfate, orlactide/glycolide copolymers, polylactide/glycolide copolymers, orethylenevinylacetate copolymers in order to control delivery of anadministered composition. For example, the oral agent can be entrappedin microcapsules prepared by coacervation techniques or by interfacialpolymerization, by the use of hydroxymethylcellulose orgelatin-microcapsules or poly (methylmethacrolate) microcapsules,respectively, or in a colloid drug delivery system. Colloidal dispersionsystems include macromolecule complexes, nano-capsules, microspheres,microbeads, and lipid-based systems, including oil-in-water emulsions,micelles, mixed micelles, and liposomes. Methods for preparation of suchformulations are known to those skilled in the art and are commerciallyavailable.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate, kaolin ormicrocrystalline cellulose, or as soft gelatin capsules wherein theactive ingredient is mixed with water or an oil medium, for examplepeanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture thereof. Such excipients aresuspending agents, for example sodium carboxymethylcellulose,methylcellulose, hydroxy-propylmethylcellulose, sodium alginate,polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing orwetting agents may be a naturally-occurring phosphatide, for examplelecithin, or condensation products of an alkylene oxide with fattyacids, for example polyoxy-ethylene stearate, or condensation productsof ethylene oxide with long chain aliphatic alcohols, for exampleheptadecaethyleneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides, for example polyethylene sorbitan monooleate. The aqueoussuspensions may also contain one or more preservatives.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified herein.

Pharmaceutical compositions of the invention may also be in the form ofoil-in-water emulsions. The oily phase may be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example, liquidparaffin, or mixtures of these. Suitable emulsifying agents may benaturally-occurring gums, for example, gum acacia or gum tragacanth;naturally-occurring phosphatides, for example, soy bean, lecithin, andesters or partial esters derived from fatty acids; hexitol anhydrides,for example, sorbitan monooleate; and condensation products of partialesters with ethylene oxide, for example, polyoxyethylene sorbitanmonooleate.

Pharmaceutical compositions can also include carriers to protect thecomposition against rapid degradation or elimination from the body, suchas a controlled release formulation, including implants, liposomes,hydrogels, prodrugs and microencapsulated delivery systems. For example,a time delay material such as glyceryl monostearate or glyceryl stearatealone, or in combination with a wax, may be employed. Prolongedabsorption of injectable pharmaceutical compositions can be achieved byincluding an agent that delays absorption, for example, aluminummonostearate or gelatin. Prevention of the action of microorganisms canbe achieved by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and thelike.

The invention also includes invention peptides in the form ofsuppositories for rectal administration. The suppositories can beprepared by mixing an invention peptide with a suitable non-irritatingexcipient which is solid at ordinary temperatures but liquid at therectal temperature and will therefore melt in the rectum to release thedrug. Such materials include, but are not limited to, cocoa butter andpolyethylene glycols.

In accordance with the invention, there are provided methods ofidentifying a peptide (or a subsequence, variant or modified form as setforth herein) that modulates bile acid homeostasis without havingsubstantial HCC activity. In one embodiment, a method includes:providing a candidate peptide sequence; administering the candidatepeptide sequence to a test animal; measuring bile acid levels of theanimal after administration of the candidate peptide sequence, todetermine if the candidate peptide sequence modulates bile acidhomeostasis; and analyzing the candidate peptide sequence for inductionof HCC in the animal, or expression of a marker correlating with HCCactivity. A candidate peptide that modulates bile acid homeostasis butdoes not have substantial HCC activity thereby identifies a peptidesequence having that modulates bile acid homeostasis without substantialHCC activity.

The terms “assaying” and “measuring” and grammatical variations thereofare used interchangeably herein and refer to either qualitative orquantitative determinations, or both qualitative and quantitativedeterminations. When the terms are used in reference to detection, anymeans of assessing the relative amount is contemplated, including thevarious methods set forth herein and known in the art. For example, bileacids and precursors, such as 7 alpha-hydroxy-4-cholesten-3-one, can beassayed or measured in a sample (e.g., serum) from a subject. Anothernon-limiting examples is a two reaction method (Randox Laboratories,Ltd.) using serum or heparinized plasma. In the first reaction bileacids are oxidized by 3-α-hydroxysteroid dehydrogenase with thesubsequent reduction of Thio-NAD to Thio-NADH. In the second reaction,oxidized bile acids are reduced by the same enzyme with the subsequentoxidation of NADH to NAD. The rate of formation of Thio-NADH isdetermined by measuring the specific absorbance change at 405 nm.

Risk factors for HCC, the most common type of liver cancer, include type2 diabetes (probably exacerbated by obesity). The risk of HCC in type 2diabetics is greater (from ˜2.5 to ˜7 times the non-diabetic risk)depending on the duration of diabetes and treatment protocol.

Various methodologies can be used in the screening and diagnosis of HCCand are well known to the skilled artisan. Indicators for HCC includedetection of a tumor maker such as elevated alpha-fetoprotein (AFP) ordes-gamma carboxyprothrombin (DCP) levels. A number of differentscanning and imaging techniques are also helpful, including ultrasound,CT scans and MRI. In relation to the invention, evaluation of whether apeptide (e.g., a candidate peptide) exhibits evidence of inducing HCCmay be determined in vivo by, for example, quantifying HCC noduleformation in an animal model, such as db/db mice, administered apeptide, compared to HCC nodule formation by wild type FGF19.Macroscopically, liver cancer may be nodular, where the tumor nodules(which are round-to-oval, grey or green, well circumscribed but notencapsulated) appear as either one large mass or multiple smallermasses. Alternatively, HCC may be present as an infiltrative tumor whichis diffuse and poorly circumscribed and frequently infiltrates theportal veins.

Pathological assessment of hepatic tissue samples is generally performedafter the results of one or more of the aforementioned techniquesindicate the likely presence of HCC. Thus, methods of the invention mayfurther include assessing a hepatic tissue sample from an in vivo animalmodel (e.g., a db/db mouse) useful in HCC studies in order to determinewhether a peptide sequence exhibits evidence of inducing HCC. Bymicroscopic assessment, a pathologist can determine whether one of thefour general architectural and cytological types (patterns) of HCC arepresent (i.e., fibrolamellar, pseudoglandular (adenoid), pleomorphic(giant cell) and clear cell).

The invention also includes the generation and use of antibodies, andfragments thereof, that bind the peptide sequences of the invention,including subsequences, sequence variants and modified forms of theexemplified peptide sequences (including the peptides listed in Tables1-10 and the Sequence Listing).

As used herein, the terms “antibodies” (Abs) and “immunoglobulins” (Igs)refer to glycoproteins having the same structural characteristics. Whileantibodies exhibit binding specificity to an antigen, immunoglobulinsinclude both antibodies and other antibody-like molecules which may lackantigen specificity.

The term “antibody” includes intact monoclonal antibodies, polyclonalantibodies, multispecific antibodies (e.g., bispecific antibodies)formed from at least two intact antibodies, and antibody bindingfragments including Fab and F(ab)′₂, provided that they exhibit thedesired biological activity. The basic antibody structural unitcomprises a tetramer, and each tetramer is composed of two identicalpairs of polypeptide chains, each pair having one “light” chain (about25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminalportion of each chain includes a variable region of about 100 to 110 ormore amino acids primarily responsible for antigen recognition. Incontrast, the carboxy-terminal portion of each chain defines a constantregion primarily responsible for effector function. Human light chainsare classified as kappa and lambda light chains, whereas human heavychains are classified as mu, delta, gamma, alpha, or epsilon, and definethe antibody's isotype as IgM, IgD, IgA, and IgE, respectively. Bindingfragments are produced by recombinant DNA techniques, or by enzymatic orchemical cleavage of intact antibodies. Binding fragments include Fab,Fab′, F(ab′)₂, Fv, and single-chain antibodies.

Each heavy chain has at one end a variable domain (VH) followed by anumber of constant domains. Each light chain has a variable domain atone end (VL) and a constant domain at its other end; the constant domainof the light chain is aligned with the first constant domain of theheavy chain, and the light chain variable domain is aligned with thevariable domain of the heavy chain. Within light and heavy chains, thevariable and constant regions are joined by a “J” region of about 12 ormore amino acids, with the heavy chain also including a “D” region ofabout 10 more amino acids. The antibody chains all exhibit the samegeneral structure of relatively conserved framework regions (FR) joinedby three hyper-variable regions, also called complementarity-determiningregions or CDRs. The CDRs from the two chains of each pair are alignedby the framework regions, enabling binding to a specific epitope. FromN-terminal to C-terminal, both light and heavy chains comprise thedomains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

An intact antibody has two binding sites and, except in bifunctional orbispecific antibodies, the two binding sites are the same. A bispecificor bifunctional antibody is an artificial hybrid antibody having twodifferent heavy/light chain pairs and two different binding sites.Bispecific antibodies can be produced by a variety of methods includingfusion of hybridomas or linking of Fab′ fragments.

As used herein, the term “monoclonal antibody” refers to an antibodyobtained from a population of substantially homogeneous antibodies, thatis, the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. In contrast to polyclonal antibodypreparations which include different antibodies directed againstdifferent determinants (epitopes), each monoclonal antibody is directedagainst a single determinant on the antigen.

A “neutralizing antibody” is an antibody molecule that is able toeliminate or significantly reduce an effector function of a targetantigen to which it binds.

Antibody binding fragments may be produced by enzymatic or chemicalcleavage of intact antibodies. Digestion of antibodies with the enzymepapain results in two identical antigen-binding fragments, also known as“Fab” fragments, and an “Fc” fragment which has no antigen-bindingactivity. Digestion of antibodies with the enzyme pepsin results in aF(ab′)₂ fragment in which the two arms of the antibody molecule remainlinked and comprise two-antigen binding sites. The F(ab′)₂ fragment hasthe ability to crosslink antigen.

The term “Fab” refers to a fragment of an antibody that comprises theconstant domain of the light chain and the CH1 domain of the heavychain. The term “Fv” when used herein refers to the minimum fragment ofan antibody that retains both antigen-recognition and antigen-bindingsites. In a two-chain Fv species, this region consists of a dimer of oneheavy-chain and one light-chain variable domain in non-covalentassociation. In a single-chain Fv species, one heavy-chain and onelight-chain variable domain can be covalently linked by a flexiblepeptide linker such that the light and heavy chains can associate in a“dimeric” structure analogous to that in a two-chain Fv species. It isin this configuration that the three CDRs of each variable domaininteract to define an antigen-binding site on the surface of the VH-VLdimer. While the six CDRs, collectively, confer antigen-bindingspecificity to the antibody, even a single variable domain (or half ofan Fv comprising only three CDRs specific for an antigen) has theability to recognize and bind antigen.

The term “complementarity determining regions” or “CDRs” refers to partsof immunological receptors that make contact with a specific ligand anddetermine its specificity. The term “hypervariable region” refers to theamino acid residues of an antibody which are responsible forantigen-binding. The hypervariable region generally comprises amino acidresidues from a “complementarity determining region” or “CDR” and/orthose residues from a “hypervariable loop”.

As used herein, the term “epitope” refers to binding sites forantibodies on protein antigens. Epitopic determinants usually consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains, as well as specific three dimensional structural andcharge characteristics. An antibody is said to bind an antigen when thedissociation constant is ≤1 μM, preferably ≤100 nM, and most preferably≤10 nM. An increased equilibrium constant (“K_(D)”) means that there isless affinity between the epitope and the antibody, whereas a decreasedequilibrium constant means that there is a higher affinity between theepitope and the antibody. An antibody with a K_(D) of “no more than” acertain amount means that the antibody will bind to the epitope with thegiven K_(D) or more strongly. Whereas K_(D) describes the bindingcharacteristics of an epitope and an antibody, “potency” describes theeffectiveness of the antibody itself for a function of the antibody.There is not necessarily a correlation between an equilibrium constantand potency; thus, for example, a relatively low K_(D) does notautomatically mean a high potency.

The term “selectively binds” in reference to an antibody does not meanthat the antibody only binds to a single substance, but rather that theK_(D) of the antibody to a first substance is less than the K_(D) of theantibody to a second substance. An antibody that exclusively binds to anepitope only binds to that single epitope.

When administered to humans, antibodies that contain rodent (murine orrat) variable and/or constant regions are sometimes associated with, forexample, rapid clearance from the body or the generation of an immuneresponse by the body against the antibody. In order to avoid theutilization of rodent-derived antibodies, fully human antibodies can begenerated through the introduction of human antibody function into arodent so that the rodent produces fully human antibodies. Unlessspecifically identified herein, “human” and “fully human” antibodies canbe used interchangeably herein. The term “fully human” can be usefulwhen distinguishing antibodies that are only partially human from thosethat are completely, or fully human. The skilled artisan is aware ofvarious methods of generating fully human antibodies.

In order to address possible human anti-mouse antibody responses,chimeric or otherwise humanized antibodies can be utilized. Chimericantibodies have a human constant region and a murine variable region,and, as such, human anti-chimeric antibody responses may be observed insome patients. Therefore, it is advantageous to provide fully humanantibodies against multimeric enzymes in order to avoid possible humananti-mouse antibody or human anti-chimeric antibody responses.

Fully human monoclonal antibodies can be prepared, for example, by thegeneration of hybridoma cell lines by techniques known to the skilledartisan. Other preparation methods involve the use of sequences encodingparticular antibodies for transformation of a suitable mammalian hostcell, such as a CHO cell. Transformation can be by any known method forintroducing polynucleotides into a host cell, including, for example,packaging the polynucleotide in a virus (or into a viral vector) andtransducing a host cell with the virus (or vector) or by transfectionprocedures known in the art. Methods for introducing heterologouspolynucleotides into mammalian cells are well known in the art andinclude dextran-mediated transfection, calcium phosphate precipitation,polybrene-mediated transfection, protoplast fusion, electroporation,encapsulation of the polynucleotide(s) in liposomes, and directmicroinjection of the DNA into nuclei. Mammalian cell lines available ashosts for expression are well known in the art and include, but are notlimited to CHO cells, HeLa cells, and human hepatocellular carcinomacells.

Antibodies can be used diagnostically and/or therapeutically. Forexample, the antibodies can be used as a diagnostic by detecting thelevel of one or more peptides of the invention in a subject, and eithercomparing the detected level to standard control level or to a baselinelevel in a subject determined previously (e.g., prior to any illness).The antibodies can be used as a therapeutic to modulate the activity ofone or more peptides of the invention, thereby having an effect on acondition or disorder.

The invention provides kits including, but not limited to, peptidesequences of the invention, optionally in combination with one or moretherapeutic agents, compositions and pharmaceutical compositionsthereof, packaged into suitable packaging material. A kit optionallyincludes a label or packaging insert including a description of thecomponents or instructions for use in vitro, in vivo, or ex vivo, of thecomponents therein. Exemplary instructions include instructions fortreatment of a bile acid related or associated disorder, such asmetabolic syndrome; a lipid- or glucose-related disorder; cholesterol ortriglyceride metabolism; type 2 diabetes; cholestasis, including, forexample diseases of intrahepatic cholestasis (e.g., PBC, PFIC, PSC, PIC,neonatal cholestasis, and drug induced cholestasis (e.g., estrogen)),and diseases of extrahepatic cholestasis (e.g., bile cut compressionfrom tumor, bile duct blockade by gall stones); bile acid malabsorptionand other disorders involving the distal small intestine, includingileal resection, inflammatory bowel diseases (e.g., Crohn's disease andulcerative colitis), disorders impairing absorption of bile acids nototherwise characterized (idiopathic)) leading to diarrhea (e.g., BAD)and GI symptoms, and GI, liver, and/or biliary cancers (e.g., coloncancer and hepatocellular cancer); and/or bile acid synthesisabnormalities, such as those contributing to NASH, cirrhosis and portalhypertension, etc.

A kit can contain a collection of such components, e.g., two or morepeptide sequences alone, or a combination of a peptide sequence withanother therapeutically useful composition (e.g., a bile acidhomeostasis modulating drug).

The term “packaging material” refers to a physical structure housing thecomponents of the kit. The packaging material can maintain thecomponents sterilely, and can be made of material commonly used for suchpurposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules,vials, tubes, etc.).

Kits of the invention can include labels or inserts. Labels or insertsinclude “printed matter,” e.g., paper or cardboard, separate or affixedto a component, a kit or packing material (e.g., a box), or attached to,for example, an ampule, tube or vial containing a kit component. Labelsor inserts can additionally include a computer readable medium, such asa disk (e.g., hard disk, card, memory disk), optical disk such as CD- orDVD-ROM/RAM, DVD, MP3, magnetic tape, or an electrical storage mediasuch as RAM and ROM or hybrids of these such as magnetic/optical storagemedia, FLASH media or memory type cards.

Labels or inserts can include identifying information of one or morecomponents therein, dose amounts, clinical pharmacology of the activeingredient(s) including mechanism of action, pharmacokinetics andpharmacodynamics. Labels or inserts can include information identifyingmanufacturer information, lot numbers, manufacturer location and date.

Labels or inserts can include information on a condition, disorder,disease or symptom for which a kit component may be used. Labels orinserts can include instructions for the clinician or for a subject forusing one or more of the kit components in a method, treatment protocolor therapeutic regimen. Instructions can include dosage amounts,frequency or duration, and instructions for practicing any of themethods, treatment protocols or therapeutic regimes set forth herein.Exemplary instructions include instructions for treatment or use of apeptide sequence as set forth herein. Kits of the invention thereforecan additionally include labels or instructions for practicing any ofthe methods and uses of the invention described herein includingtreatment methods and uses.

Labels or inserts can include information on any benefit that acomponent may provide, such as a prophylactic or therapeutic benefit.Labels or inserts can include information on potential adverse sideeffects, such as warnings to the subject or clinician regardingsituations where it would not be appropriate to use a particularcomposition. Adverse side effects could also occur when the subject has,will be or is currently taking one or more other medications that may beincompatible with the composition, or the subject has, will be or iscurrently undergoing another treatment protocol or therapeutic regimenwhich would be incompatible with the composition and, therefore,instructions could include information regarding such incompatibilities.

Invention kits can additionally include other components. Each componentof the kit can be enclosed within an individual container and all of thevarious containers can be within a single package. Invention kits can bedesigned for cold storage. Invention kits can further be designed tocontain peptide sequences of the invention, or that contain nucleicacids encoding peptide sequences. The cells in the kit can be maintainedunder appropriate storage conditions until ready to use.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed herein.

All applications, publications, patents and other references, GenBankcitations and ATCC citations cited herein are incorporated by referencein their entirety. In case of conflict, the specification, includingdefinitions, will control. As used herein, the singular forms “a”,“and,” and “the” include plural referents unless the context clearlyindicates otherwise. Thus, for example, reference to “a peptidesequence” or a “treatment,” includes a plurality of such sequences,treatments, and so forth.

As used herein, numerical values are often presented in a range formatthroughout this document. The use of a range format is merely forconvenience and brevity and should not be construed as an inflexiblelimitation on the scope of the invention unless the context clearlyindicates otherwise. Accordingly, the use of a range expressly includesall possible subranges, all individual numerical values within thatrange, and all numerical values or numerical ranges including integerswithin such ranges and fractions of the values or the integers withinranges unless the context clearly indicates otherwise. This constructionapplies regardless of the breadth of the range and in all contextsthroughout this patent document. Thus, for example, reference to a rangeof 90-100% includes 91-99%, 92-98%, 93-95%, 91-98%, 91-97%, 91-96%,91-95%, 91-94%, 91-93%, and so forth. Reference to a range of 90-100%also includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%,91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%,etc., and so forth.

In addition, reference to a range of 1-3, 3-5, 5-10, 10-20, 20-30,30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120,120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190, 190-200,200-225, 225-250 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, etc. In a further example, reference to a rangeof 25-250, 250-500, 500-1000, 1000-2500 or 2500-5000, 5000-25,000,5000-50,000 includes any numerical value or range within or encompassingsuch values, e.g., 25, 26, 27, 28, 29 . . . 250, 251, 252, 253, 254 . .. 500, 501, 502, 503, 504 . . . , etc.

As also used herein a series of ranges are disclosed throughout thisdocument. The use of a series of ranges include combinations of theupper and lower ranges to provide another range. This constructionapplies regardless of the breadth of the range and in all contextsthroughout this patent document. Thus, for example, reference to aseries of ranges such as 5-10, 10-20, 20-30, 30-40, 40-50, 50-75,75-100, 100-150, includes ranges such as 5-20, 5-30, 5-40, 5-50, 5-75,5-100, 5-150, and 10-30, 10-40, 10-50, 10-75, 10-100, 10-150, and 20-40,20-50, 20-75, 20-100, 20-150, and so forth.

For the sake of conciseness, certain abbreviations are used herein. Oneexample is the single letter abbreviation to represent amino acidresidues. The amino acids and their corresponding three letter andsingle letter abbreviations are as follows:

alanine Ala (A) arginine Arg (R) asparagine Asn (N) aspartic acid Asp(D) cysteine Cys (C) glutamic acid Glu (E) glutamine Gln (Q) glycine Gly(G) histidine His (H) isoleucine Ile (I) leucine Leu (L) lysine Lys (K)methionine Met (M) phenylalanine Phe (F) proline Pro (P) serine Ser (S)threonine Thr (T) tryptophan Trp (W) tyrosine Tyr (Y) valine Val (V)

The invention is generally disclosed herein using affirmative languageto describe the numerous embodiments. The invention also specificallyincludes embodiments in which particular subject matter is excluded, infull or in part, such as substances or materials, method steps andconditions, protocols, procedures, assays or analysis. Thus, even thoughthe invention is generally not expressed herein in terms of what theinvention does not include, aspects that are not expressly included inthe invention are nevertheless disclosed herein.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, the following examples are intended to illustrate but notlimit the scope of invention described in the claims.

EXAMPLES Example 1

The following is a description of various methods and materials used inthe studies herein.

Animals.

db/db mice were purchased from The Jackson Laboratory (Bar Harbor, Me.),Mice were kept in accordance with welfare guidelines under controlledlight (12 hr light and 12 hr dark cycle, dark 6:30 pm-6:30 am),temperature (22±4° C.) and humidity (50%±20%) conditions. Mice had freeaccess to water (autoclaved distilled water) and were fed ad libitum ona commercial diet (Harlan Laboratories, Indianapolis, Ind., Irradiated2018 Teklad Global 18% Protein Rodent Diet) containing 17 kcal % fat, 23kcal % protein and 60 kcal % carbohydrate. All animal studies wereapproved by the NGM Institutional Animal Care and Use Committee.

DNA and Amino Acid Sequences.

cDNA of ORF encoding human FGF19 (Homo sapiens FGF19, GenBank AccessionNo. NM_005117.2) variants. Protein sequence encoded by the cDNA (GenBankAccession No. NP_005108.1).

PCR.

FGF19 ORF was amplified with polymerase chain reaction (PCR) usingrecombinant DNA (cDNA) prepared from human small intestinal tissue. PCRreagents kits with Phusion® high-fidelity DNA polymerase were purchasedfrom New England BioLabs (F-530L, Ipswich, Mass.). The following primerswere used: forward PCR primer:

(SEQ ID NO: 136) 5′ CCGACTAGTCACCatgcggagcgggtgtgtggand reverse PCR primer:

(SEQ ID NO: 137) 5′ ATAAGAATGCGGCCGCTTACTTCTCAAAGCTGGGACTCCTC.Amplified DNA fragment was digested with restriction enzymes Spe I andNot I (the restriction sites were included in the 5′ or 3′ PCR primers,respectively) and was then ligated with AAV transgene vectors that hadbeen digested with the same restriction enzymes. The vector used forexpression contained a selectable marker and an expression cassettecomposed of a strong eukaryotic promoter 5′ of a site for insertion ofthe cloned coding sequence, followed by a 3′ untranslated region andbovine growth hormone polyadenylation tail. The expression construct isalso flanked by internal terminal repeats at the 5′ and 3′ ends.

Cyp7a1 Repression Assay in Primary Human Hepatocytes.

Primary human hepatocytes were plated on collagen coated plates (BectonDickinson Biosciences) in Williams E media (Invitrogen) supplementedwith 100 nM dexamethasone (Sigma) and 0.25 mg/ml MatriGel™ (BectonDickinson Biosciences). Cells were treated with FGF19 or variants at 37°C. for 6 hours. Cyp7a1 expression was evaluated in triplicate byquantitative RT-PCR (TaqMan® ABI PRISM 7700, Applied Biosystems) andnormalized to GAPDH expression.

Cyp7a1 In Vivo Repression Assay.

Nine-week-old male db/db mice (Jackson Laboratories) were injectedintraperitoneally with recombinant proteins FGF19 or FGF21 at 0.1 mg/kg,1 mg/kg, and 10 mg/kg. Animals were euthanized 5 hours post-injection.Liver was harvested and homogenized in TRIzol® reagent (Invitrogen).Total RNA was extracted and treated with DNase (Ambion) followed byquantitative RT-PCR analysis and normalized to GAPDH expression.

Production and Purification of AAV.

AAV293 cells (obtained from Agilent Technologies, Santa Clara, Calif.)were cultured in Dulbeco's Modification of Eagle's Medium (DMEM,Mediatech, Inc. Manassas, Va.) supplemented with 10% fetal bovine serumand 1× antibiotic-antimycotic solution (Mediatech, Inc. Manassas, Va.).The cells were plated at 50% density on day 1 in 150 mm cell cultureplates and transfected on day 2, using calcium phosphate precipitationmethod with the following 3 plasmids (20 μg/plate of each): AAVtransgene plasmid, pHelper™ plasmids (Agilent Technologies) and AAV2/9plasmid (Gao et al., J. Virol. 78:6381 (2004)). Forty-eight (48) hoursafter transfection, the cells were scraped off the plates, pelleted bycentrifugation at 3000×g and resuspended in buffer containing 20 mM TrispH 8.5, 100 mM NaCl and 1 mM MgCl₂. The suspension was frozen in analcohol dry ice bath and was then thawed in 37° C. water bath. Thefreeze and thaw cycles were repeated three times; Benzonase®(Sigma-aldrich, St. Louis, Mo.) was added to 50 units/ml; deoxycholatewas added to a final concentration of 0.25%. After an incubation at 37°C. for 30 min, cell debris was pelleted by centrifugation at 5000×g for20 min. Viral particles in the supernatant were purified using adiscontinued iodixanal (Sigma-aldrich, St. Louis, Mo.) gradient aspreviously described (Zolotukhin S. et al (1999) Gene Ther. 6:973). Theviral stock was concentrated using Vivaspin® 20 (MW cutoff 100,000Dalton, Sartorius Stedim Biotech, Aubagne, France) and re-suspended inphosphate-buffered saline (PBS) with 10% glycerol and stored at −80° C.To determine the viral genome copy number, 2 μl of viral stock wereincubated in 6 μl of solution containing 50 units/ml Benzonase®, 50 mMTris-HCl pH 7.5, 10 mM MgCl₂ and 10 mM CaCl₂ at 37° C. for 30 minutes.

Afterwards, 15 μl of the solution containing 2 mg/ml of Proteinase K,0.5% SDS and 25 mM EDTA were added and the mixture was incubated foradditional 20 min at 55° C. to release viral DNA. Viral DNA was cleanedwith mini DNeasy® Kit (Qiagen, Valencia, Calif.) and eluted with 40 μlof water. Viral genome copy (GC) was determined by using quantitativePCR.

Viral stock was diluted with PBS to desirable GC/ml. Viral workingsolution (200 μl) was delivered into mice via tail vein injection.

Hepatocellular Carcinoma (HCC) Assay.

Liver specimens were harvested from db/db mice 24 weeks after AAVinjection. HCC scores were recorded as the number of HCC nodules on thesurface of the entire liver from variants-injected mice divided by thenumber of HCC nodules from wild-type FGF19-injected mice.

Serum FGF19/FGF21/Variants Exposure Level Assay.

Whole blood (about 50 μl/mouse) from mouse tail snips was collected intoplain capillary tubes (BD Clay Adams SurePrep™, Becton Dickenson and Co.Sparks, Md.). Serum and blood cells were separated by spinning the tubesin an Autocrit™ Ultra 3 (Becton Dickinson and Co. Sparks, Md.). FGF19,FGF21, and variant exposure levels in serum was determined using EIAkits (Biovendor) by following the manufacturer's instructions.

FGFR4 Binding and Activity Assays.

Solid phase ELISA (binding) and ERK phosphorylation assay can beperformed using purified recombinant proteins. FGFR binding assay can beconducted using solid phase ELISA. Briefly, a 96-well plate can becoated with 2 μg/ml anti-hFc antibody and can be incubated with 1 μg/mlFGFR1-hFc or FGFR4-hFc. Binding to FGF19 variants in the presence of 1μg/ml soluble β-klotho and 20 μg/ml heparin can be detected bybiotinylated anti-FGF19 antibodies (0.2 μg/mL), followed bystreptavidin-HRP incubation (100 ng/mL). For FGFR4 activation assay,Hep3B cells can be stimulated with FGF19 variants for 10 minutes at 37°C., then can be immediately lysed and assayed for ERK phosphorylationusing a commercially available kit from Cis-Bio.

Example 2

In order to confirm that FGF19 variants such as those set forth hereinrepress cyp7a1 expression, inhibition of cyp7a1 expression by wild-typeFGF19 was determined following administration of various concentrations.The effects of FGF21 were assessed in a comparable manner.

Briefly, at time 0, db/db mice were dosed intraperitoneally with eitherrecombinant FGF19 (0.1 mg/kg; 1 mg/kg; 10 mg/kg) or recombinant FGF21(0.1 mg/kg; 1 mg/kg; 10 mg/kg). Five hours after dosing, livers wereharvested, RNA was extracted, and cyp7a1 expression was determined byreal-time PCR (QPCR) using GADPH as a normalization control. In eachgroup of mice, n=3, and cyp7a1 expression values for the various FGF19and FGF21 concentrations were compared to mice dosed with PBS vehiclecontrol.

As set forth in FIG. 1, FGF19 dramatically decreased cyp7a1 expressionin a concentration-dependent manner. Although administration of FGF21caused a reduction of cyp7a1 expression, the effect was demonstrablyless than that observed with FGF19.

The effect of variant M70 on cyp7a1 expression in human primaryhepatocytes was compared to that of FGF19. As noted in FIG. 2, variantM70 repressed cyp7a1 expression in an amount comparable to that ofFGF19.

Example 3

Using the assays described above, repression of cyp7a1 in primary humanhepatocytes was determined for a number of FGF19 variants. As indicatedin FIG. 3-FIG. 5, several variants (e.g., M1, M2, etc.) exhibited strongcyp7a1 repression.

To evaluate effects of some additional FGF19 variants on Cyp7a1repression, the in vitro cell-based assay (primary human hepatocyte) andthe in vivo assay (protein dosing in db/db mice) were utilized in whichthe variants were compared with saline-treated controls. FIG. 5 setsforth the results (IC₅₀ and Cyp7a1(%)) in tabular form. While most FGF19variants that were evaluated exhibited Cyp7a1-inhibiting activity, a fewvariants (e.g., M90, M96, M98, M5 and M32) no longer repressed Cyp7a1.

FGF19 variants that retain Cyp7a1 repression activity can be furtherevaluated in the HCC assay (or other relevant assay or model) describedabove to identify variants that might be useful for modulating bile acidmetabolism and/or for treating bile acid-related diseases (e.g., bileacid diarrhea and primary biliary cirrhosis) without causing inductionof HCC. The figures set forth data for variants that were evaluated inthe HCC assay.

Example 4

The following is a data summary of 25 additional variant peptidesanalyzed for lipid elevating activity and tumorigenesis. The dataclearly show a positive correlation between lipid elevation andtumorigenesis, as determined by HCC formation in db/db mice.

The Tables summarize different variant peptides. Such exemplifiedvariant peptides have FGF19 C-terminal sequence:PHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ ID NO:188) at theC-terminal portion, e.g., following the “TSG” amino acid residues.Notably, variant peptides (7 total, including M5) that did not cause astatistically significant elevation of lipids did not induce HCCformation. In contrast, all variant peptides (17 total) that caused astatistically significant elevation of lipids also caused HCC formationin mice. This data indicates that there is a strong positive correlationbetween lipid elevating activity and HCC formation. Accordingly, lipidelevating activity can be used as an indicator and/or predictor of HCCformation in animals.

TABLE 1Elevated Triglyceride and Cholesterol in db/db Mice Appears to PositivelyCorrelate With HCC Formation (see SEQ ID NOs: 99, 5 and 74 to 81).

      SEQ ID NO.       Core   SEQ ID NO.   Lipid Eleva- tion   HCCForma- tion FGF19 RPLAFSDAGPHVHYGWGDPI  99 (aa 1-20) RLRHLYTSG 185 + +FGF21 HPIPDSSPLLQ--FGGQV 100 (aa 1-16) RQRYLYTDD 186 − − M5R-HPIPDSSPLLQ--FGGQV   5 (aa 1-17) RLRGLYTSG 185 − − M74R ------------------DAGPHVHYGWGDPI  74 (aa 1-15) RLRGLYTSG 185 + + M75R --------------------------- VHYGWGDPI  75 (aa 1-10) RLRGLYTSG 185 − −M76 R -------------------------------------- GDPI  76 (aa 1-5) RLRGLYTSG185 − − M77 R -----------------------------------------------  77 (aa 1)RLRGLYTSG 185 − − M78 R ------------------- AGPHVHYGWGDPI  78 (aa 1-14)RLRGLYTSG 185 + + M79 R --------------------- GPHVHYGWGDPI  79 (aa 1-13)RLRGLYTSG 185 + + M80 R ----------------------- PHVHYGWGDPI 80 (aa 1-12) RLRGLYTSG 185 − − M81R ------------------------- HVHYGWGDPI  81 (aa 1-11) RLRGLYTSG 185 − −

TABLE 2Elevated Triglyceride and Cholesterol in db/db Mice Appears to PositivelyCorrelate with HCC Formation (see SEQ ID NOs: 99, 100 and 82 to 98).

      SEQ ID NO.       Core     SEQ ID NO.     Lipid Elevation   HCCForma- tion FGF19 RPLFSDAGPHVHYGWGDPI  99 (aa 1-20) RLRHLYTSG 185 + +FGF21 HPIPDSSPLLQ--FGGQV 100 (aa 1-16) RQRYLYTDD 186 − − M82RPLAFSAAGPHVHTGWGDPI  82 (aa 1-20) RLRGLYTSG 185 + + M83RPLAFSDAAPHVHYGWGDPI  83 (aa 1-20) RLRGLYTSG 185 +/− +/− M84RPLAFSDAGAHVHYGWGDPI  84 (aa 1-20) RLRGLYTSG 185 +/− +/− M85RPLAFSDAGPHVHYGAGDPI  85 (aa 1-20) RLRGLYTSG 185 − − M86RPLAFDAGPHVHYGWGAPI  86 (aa 1-20) RLRGLYTSG 185 + + M87RPLAFSDAGPHVHYGWGDAI  87 (aa 1-20) RLRGLYTSG 185 + +

TABLE 3Elevated Triglyceride and Cholesterol in db/db Mice Appears to PositivelyCorrelate with HCC Formation (see SEQ ID NOs: 99, 100 and 88 to 98)

      Core       SEQ ID NO     Lipid Elevation     HCC Formation FGF19RPLAFSDAGPHVHYGWGDPI RLRHLYTSG  99 (aa 1-29) + + FGF21  HPIPDSSPLLQ--FGGQV RQRYLYTDD 100 (aa 1-25) − − H31A/S141A(M88)FGF19 + + H31A/H142A(M89) FGF19 + + K127A/R129A(M90) FGF19 + +K127A/S141A(M91) FGF19 + + K127A/H142A(M92) FGF19 + + R129A/S141A(M93)FGF19 + + S141A/H142A(M94) FGF19 + + K127A/H142A(M95) FGF19 + +K127A/R129A/S141A(M96) FGF19 + + K127A/R129A/H142A(M97) FGF19 + +K127A/R129A/S141A/ FGF19 + + H142A(M98)

M88 (H31A/S141A): (SEQ ID NO: 88)RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPAGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLAHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M89 (H31A/H142A):(SEQ ID NO: 89) RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPAGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSAFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M90 (K127A/R129A):(SEQ ID NO: 90) RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAAQAQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M91 (K127A/S141A):(SEQ ID NO: 91) RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAAQRQLYKNRGFLPLAHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M92 (K127A/H142A):(SEQ ID NO: 92) RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAAQRQLYKNRGFLPLSAFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M93 (R129A/S141A):(SEQ ID NO: 93) RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQAQLYKNRGFLPLAHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M94 (S141A/H142A):(SEQ ID NO: 94) RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLAAFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M95 (K127A/H142A):(SEQ ID NO: 95) RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAAQRQLYKNRGFLPLSAFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M96 (K127A/R129A/S141A):(SEQ ID NO: 96) RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAAQAQLYKNRGFLPLAHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M97 (K127A/R129A/H142A):(SEQ ID NO: 97) RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAAQAQLYKNRGFLPLSAFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEKM98 (K127A/R129A/S141A/H142A): (SEQ ID NO: 98)RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAAQAQLYKNRGFLPLAAFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK

Example 5

The following is a data summary of additional FGF19 variant peptidesanalyzed for glucose lowering activity and lipid elevating activity.

Table 4 illustrates the peptide “core sequences” of 35 additional FGF19variants, denoted M5 to M40. Such exemplified variant peptides haveFGF19 C-terminal sequence,PHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ ID NO: 188) at theC-terminal portion, e.g., following the “TSG” amino acid residues of thecore sequence. The data clearly show that variants M6, M7, M8, mM38 andM39 have the desired characteristics of glucose lowering activity andnot statistically significant lipid elevating activity in db/db mice.

TABLE 4 Additional Variants and Fine Mapping of the N-terminal Domain(see SEQ ID NOs: 99, 100, and 5 to 40) SEQ ID NO of N-term- SEQ IDGlucose Lipid N-terminal Domain Domain Core NO. Lowering Elevation FGF19RPLAFSDAGPHVHYGWGDPI  99 (aa 1-20) RLRHLYTSG 185 + + FGF21-HPIPDSSPLLQ--FGGQV 100 (aa 1-16) RQRYLYTDD 186 + - M5RHPIPDSSPLLQ--FGGQV   5 (aa 1-17) RLRHLYTSG 185 + - M6R----DSSPLLQ--FGGQV   6 (aa 1-18) RLRHLYTSG 185 + - M7RPLAFSDSSPLLQ--FGGQV   7 (aa 1-18) RLRHLYTSG 185 + - M8R-HPIPDSSPLLQ--WGDPI   8 (aa 1-17) RLRHLYTSG 185 + - M9R-HPIPDSSPLLQFGWGDPI   9 (aa 1-19) RLRHLYTSG 185 + + M10R-HPIPDSSPHVHYGWGDPI  10 (aa 1-19) RLRHLYTSG 185 - + M11RPLAFSDAGPLLQ--WGDPI  11 (aa 1-18) RLRHLYTSG 185 N/D N/D M12RPLAFSDAGPLLQFGWGDPI  12 (aa 1-20) RLRHLYTSG 185 - + M13RPLAFSDAGPLLQ--FGGQV  13 (aa 1-18) RLRHLYTSG 185 - - M14R-HPIPDSSPHVHYG--GQV  14 (aa 1-17) RLRHLYTSG 185 - - M15RPLAFSDAGPHVHYG--GQV  15 (aa 1-18) RLRHLYTSG 185 + + M16RPLAFSDAGPHVH--WGDPI  16 (aa 1-18) RLRHLYTSG 185 N/D N/D M17RPLAFSDAGPHV--GWGDPI  17 (aa 1-18) RLRHLYTSG 185 N/D N/D M18RPLAFSDAGPH--YGWGDPI  18 (aa 1-18) RLRHLYTSG 185 N/D N/D M19RPLAFSDAGP-V-YGWGDPI  19 (aa 1-18) RLRHLYTSG 185 N/D N/D M20RPLAFSDAGP-VH-GWGDPI  20 (aa 1-18) RLRHLYTSG 185 N/D N/D M21RPLAFSDAGP-VHY-WGDPI  21 (aa 1-18) RLRHLYTSG 185 N/D N/D M22RPLAFSDAGPHVH-GWGDPI  22 (aa 1-18) RLRHLYTSG 185 N/D N/D M23RPLAFSDAGPH-H-GWGDPI  23 (aa 1-18) RLRHLYTSG 185 N/D N/D M24RPLAFSDAGPH-HY-WGDPI  24 (aa 1-18) RLRHLYTSG 185 N/D N/D M25RPLAFSDAGPHV-Y-WGDPI  25 (aa 1-18) RLRHLYTSG 185 N/D N/D M26RPLAFSDSSPLVH--WGDPI  26 (aa 1-18) RLRHLYTSG 185 N/D N/D M27RPLAFSDSSPHVH--WGDPI  27 (aa 1-18) RLRHLYTSG 185 N/D N/D M28RPLAFSDAPHV----WGDPI  28 (aa 1-16) RLRHLYTSG 185 N/D N/D M29RPLAFSDAGPHVHY-WGDPI  29 (aa 1-19) RLRHLYTSG 185 N/D N/D M30RPLAFSDAGPHVHYAWGDPI  30 (aa 1-20) RLRHLYTSG 185 N/D N/D M31R-HPIPDSSPLLQ--FGAQV  31 (aa 1-17) RLRHLYTSG 185 +/- - M32R-HPIPDSSPLLQ--FGIYQV  32 (aa 1-18) RLRHLYTSG 185 - - M33R-HPIPDSSPLLQ--FGGQV  33 (aa 1-17) RLRHLYTSG 185 - - M34R-HPIPDSSPLLQ--FGGAV  34 (aa 1-17) RLRHLYTSG 185 +/- - M35R-HPIPDSSPLLQ--FGGEV  35 (aa 1-17) RLRHLYTSG 185 +/- +/ M36R-HPIPDSSPLLQ--FGGQV  36 (aa 1-17) RLRHLYTSG 185 +/- - M37R-HPIPDSSPLLQ--FGGUA  37 (aa 1-17) RLRHLYTSG 185 - - M38R-HPIPDSSPLLQ--FGGQT  38 (aa 1-17) RLRHLYTSG 185 + - M39R-HPIPDSSPLLQ--FGGQT  39 (aa 1-17) RLRHLYTSG 185 + - M40R-HPIPDSSPLLQFGWGQP  40 (aa 1-16) RLRHLYTSG 185 - +

TABLE 4a(see SEQ ID NOs: 99, 100, 5, 9, 8, 12, 10, 13, 15, 14, 43, 6 and 7)

    Core     SEQ ID NO.   Glucose Lowering   Lipid Elevation   HCCFormation FGF19 RPLAFSDAGPHVHYGWGDPI RLRHLYTSG  99 (aa 1-29) + + + FGF21  HPIPDSSPLLQ--FGGQV RQRYLYTDD 100 (aa 1-25) + − − M5R-HPIPDSSPLLQ--FGGQV RLRHLYTSG   5 (aa 1-26) + − − M9R-HPIPDSSPLLQFGWGDPI RLRHLYTSG   9 (aa 1-28) + + + M8R-HPIPDSSPLLQ--WGDPI RLRHLYTSG   8 (aa 1-26) + + + M12RPLAFSDAGPLLQFGWGDPI RLRHLYTSG  12 (aa 1-29) − + + M10R-HPIPDSSPHVHYGWGDPI RLRHLYTSG  10 (aa 1-28) − + + M13RPLAFSDAGPLLQ--FGGQV RLRHLYTSG  13 (aa 1-27) − + + M15RPLAFSDAGPVHVHYG--GQV RLRHLYTSG  15 (aa 1-27) − − +/− M14R-HPIPDSSPHVHYG--GQV RLRHLYTSG  14 (aa 1-26) − − +/− M43RPLAFSDAGPHVHYG-GD-I RLRHLYTSG  43 (aa 1-27) + − +/− M6R-----DSSPLLQ--FGGQV RLRHLYTSG   6 (aa 1-262) + − − M7RPLAFSDSSPLLQ--FGGQV RLRHLYTSG   7 (aa 1-27) − − −

TABLE 4b (see SEQ ID NOs: 99, 5 and 31 to 40)

    Core     SEQ ID NO.   Glucose Lowering   Lipid Elevation   HCCFormation FGF19 RPLAFSDAGPHVHYGWGDPI RLRHLYTSG  99 (aa 1-29) + + + FGF21  HPIPDSSPLLQ--FGGQV RQRYLYTDD 100 (aa 1-25) + − − M5R-HPIPDSSPLLQ--FGGQV RLRHLYTSG   5 (aa 1-26) + − − M31R-HPIPDSSPLLQ--FGAQV RLRHLYTSG  31 (aa 1-26) + − + M32R-HPIPDSSPLLQ--FGDQV RLRHLYTSG  32 (aa 1-26) + − − M33R-HPIPDSSPLLQ--FGPQV RLRHLYTSG  33 (aa 1-26) − − + M34R-HPIPDSSPLLQ--FGGAV RLRHLYTSG  34 (aa 1-26) − − + M35R-HPIPDSSPLLQ--FGGEV RLRHLYTSG  35 (aa 1-26) − − + M36R-HPIPDSSPLLQ--FGGNV RLRHLYTSG  36 (aa 1-26) + − +/− M37R-HPIPDSSPLLQ--FGGQA RLRHLYTSG  37 (aa 1-26) − − + M38R-HPIPDSSPLLQ--FGGQI RLRHLYTSG  38 (aa 1-26) − − + M39R-HPIPDSSPLLQ--FGGQT RLRHLYTSG  39 (aa 1-26) − − + M40R-HPIPDSSPLLQFGWGQPV RLRHLYTSG  40 (aa 1-28) − + +

TABLE 4c (see SEQ ID NOs: 99, 100, 5, 52, 54, to 68, 4, 69, 70 and 53)

    Core     SEQ ID NO.   Glucose Lowering   Lipid Elevation   HCCFormation FGF19 RPLAFSDAGPHVHYGWGDPI RLRHLYTSG  99 (aa 1-29) + + + FGF21  HPIPDSSPLLQ--FGGQV RQRYLYTDD 100 (aa 1-25) + − − M5R-HPIPDSSPLLQ--FGGQV RLRHLYTSG   5 (aa 1-26) + − − M52R-----DSSPLLQ--WGDPI RLRHLYTSG  52 (aa 1-22) + + − M54RPLAFSDAGPLLQ--WGDPI RLRHLYTSG  54 (aa 1-27) − + + M55RPLAFSDAGPH--YGWGDPI RLRHLYTSG  55 (aa 1-27) − + + M56RPLAFSDAGP-V-YGWGDPI RLRHLYTSG  56 (aa 1-27) − + + M57RPLAFSDAGP-VT-GWGDPI RLRHLYTSG  57 (aa 1-27) − + + M58RPLAFSDAGP-VHY-WGDPI RLRHLYTSG  58 (aa 1-27) − + + M59RPLAFSDAGPH-H-GWGDPI RLRHLYTSG  59 (aa 1-27) − + + M60RPLAFSDAGPH-HY-WGDPI RLRHLYTSG  60 (aa 1-27) − + + M61RPLAFSDAGPHV--GWGDPI RLRHLYTSG  61 (aa 1-27) − + + M62RPLAFSDAGPHV-Y-WGDPI RLRHLYTSG  62 (aa 1-27) − + + M63RPLAFSDAGPHVH--WGDPI RLRHLYTSG  63 (aa 1-27) + + + M64RPLAFSDSSPLVH--WGDPI RLRHLYTSG  64 (aa 1-27) + + + M65RPLAFSDSSPHVH--WGDPI RLRHLYTSG  65 (aa 1-27) − + + M66RPLAFSDAGPHLQ--WGDPI RLRHLYTSG  66 (aa 1-27) + + + M67RPLAFSDAGPHV---WGDPI RLRHLYTSG  67 (aa 1-26) − − +/− M68RPLAFSDAGPHVHY-WGDPI RLRHLYTSG  68 (aa 1-28) − + − M4RPLAFSDAGPHVHYAWGDPI RLRHLYTSG   4 (aa 1-29) + + + M69R-----DSSPLVHYGWGDPI RLRHLYTSG  69 (aa 1-24) + + − M70MR----DSSPLVHYGWGDPI RLRHLYTSG  70 (aa 1-25) + + − M53M-----DSSPLLQ--WGDPI RLRHLYTSG 192 (aa 1-22) + + −

Table 5 illustrates the peptide sequences of additional variants.

TABLE 5 Additional Variants (SEQ ID NOs: 41, 42 and 44-46) M41:(SEQ ID NO: 41) RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS M42: (SEQ ID NO: 42)HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGELPLSHFLPMLPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS M44: (SEQ ID NO: 44)RPLAFSDAGPHVHYGWGDPIRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHEWPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS M45: (SEQ ID NO: 45)HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHEWPAPRGPARFLPLPGLPPALPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M46: (SEQ ID NO: 46)RPLAFSDAGPHVHYGWGDPIRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHEWPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYASPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK

Table 6 illustrates the peptide sequences of 3 FGF19 variants, denotedM1, M2 and M69. The data clearly show that these three variants have thedesired characteristics of glucose lowering activity in db/db mice.These three variants appear to elevate lipids in db/db mice.

TABLE 6 Additional Variants (SEQ ID NOs: 1, 2 and 69) M1:(SEQ ID NO: 1 or 139) RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M2:(SEQ ID NO: 2 or 140) RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M69: (SEQ ID NO: 69)RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK

Example 6

The following is a data summary showing that FGF19 reduces body weightin diet-induced obese mice and in ob/ob mice, and liver tumor formationactivity and body weight in db/db mice.

Mice were injected with FGF19 or FGF21 in AAV vector. Body weight wasrecorded 4 weeks after injection.

TABLE 7 FGF19 reduces body weight in diet-induced obesemice and in ob/ob mice (sequences correspond to aa 1-29of SEQ ID NO: 99 and aa 1-25 of SEQ ID NO: 100, respectively)

    core   Body Weight- Lowering in DIO   Body Weight- Lowering in Ob/obFGF19 RPLAFSDAGPHVHYGWGDPI RLRHLYTSQ + + FGF21   HPIPDSSPLLQ--FGGQVRQRYLYTDD + +

TABLE 8Correlation of body weight and liver tumor formation of FGF19, FGF21 and selectedvariants in db/dbb mice (see, e.g., SEQ ID NOs: 99, 100, 5, 6, 32, 52 and 69)

    core     SEQ ID NO Liver Tumor Nodule   Body Weight FGF19RPLAFSDAGPHVHYGWGDPI RLRHLYTSG  99 (aa 1-29) + Increased FGF21  HPIPDSSPLLQ--FGGQV RQRYLYTDD 100 (aa 1-25) − Decreased M5R-HPIPDSSPLLQ--FGGQV RLRHLYTSG   5 (aa 1-26) − Increased M6R-----DSSPLLQ--FGGQV RLRHLYTSG   6 (aa 1-22) − Decreased M32R--HPIPDSSPLLQ-FGDQV RLRHLYTSG  32 (aa 1-26) − Decreased M52R-----DSSPLLQ--WGDPI RLRHLYTSG  52 (aa 1-22) − Decreased M69R-----DSSPLVHYGWGDPI RLRHLYTSG  69 (aa 1-24) − Increased

Example 7

The following is a study showing that variant M5 and variant M69peptides reduce blood glucose.

Mice (ob/ob) were injected (subcutaneously) with M5 (0.1 and 1 mg/kg,s.c.) or FGF19 (1 mg/kg, s.c.), or variant M69 (0.1 and 1 mg/kg, s.c.)or FGF19 (1 mg/kg, s.c.). Plasma glucose levels were measured at 2, 4,7, and 24 hours after injection. The results of variant M5 and variantM69 showed similar glucose lowering effects as wild type FGF19 (data notshown).

Example 8

This example sets forth several variant polypeptides and particularcharacteristics thereof, including the variants' effect on glucoselowering, lipid profile parameters, and HCC formation.

In particular, Table 9 compares data generated for variants M5 (SEQ IDNO:5), M6 (SEQ ID NO:6) and M50 (SEQ ID NO:50) with data generated forcorresponding variant polypeptides (denoted as M144, M145, and M146,respectively) having N-terminal Arg (R) deletions. Only certain sequencedomains for each variant are listed: N-terminal domain, Core, andSheet-8/Loop-8/Sheet-9 region.

TABLE 9

      Core Sheet- 8/Loop8/ Sheet-9 region Glu- cose Lower- ing BodyWeight Re- duction   HDL Eleva- tion   Tri- glyceride Elevation   HCCForma- tion FGF19 RPLAFSDAGPHVHYGWGDPI RLRHLYTSG //EEIRPDGYNVY// +− + + + (aa 1-20 of SEQ ID (aa 21-29 of (aa 102-112 of NO: 99)SEQ ID NO: 99) SEQ ID NO: 99) FGF21 HPIPDSSPLLQ--FGGQV RQRYLYTDD//ELLLEDGYNVY// + + − − − (aa 1-20 of SEQ ID (aa 21-29 of (aa 97-107 ofNO: 100) SEQ ID SEQ ID NO: 100) NO: 100) M5 R-HPIPDSSPLLQ--FGGQVRLRHLYTSG //EEIRPDGYNVY// + − − − − (aa 1-17 of SEQ ID (aa 18-26 of(aa 99-109 of NO: 5) SEQ ID NO: 5) SEQ ID NO: 5) M6R-------DSSPLLQ-FGGQV RLRHLYTSG //EEIRPDGYNVY// + − − − −(aa 1-14 of SEQ ID (aa 15-23 of (aa 95-105 of NO: 6) SEQ ID NO: 6)SEQ ID NO: 6) M50 R-HPIPDSSPLLQ-FGDQV RLRHLYTSG //EEIRPDGYNVY// + + − −− (aa 1-17 of SEQ ID (aa 18-26 of (ss 99-109 of NO: 50) SEQ ID NO: 50)SEQ ID NO: 50) M144 --HPIPDSSPLLQ--FGGQV RLRHLYTSG //EEIRPDGYNVY// + − −− − (aa 2-17 of SEQ ID (aa 18-26 of (aa 99-109 of NO: 5) SEQ ID NO: 5)SEQ ID NO: 5) M145 -----DSSPLLQ--FGGQV RLRHLYTSG //EEIRPDGYNVY// + − − −− (aa 2-14 of SEQ ID (aa 15-23 of (aa 95-105 of NO: 6) SEQ ID NO: 6)SEQ ID NO: 6) M146 --HPIPDSSPLLQ--FGDQV RLRHLYTSG //EEIRPDGYNVY// + + −− − (aa 2-17 of SEQ ID (aa 18-26 of (aa 99-109 of NO: 50) SEQ ID NO: 50)SEQ ID NO: 50)

As the data in Table 9 indicate, the deletion of the N-terminal Arg (R)did not significantly impact glucose lowering, body weight reduction,HDL and triglyceride elevation, and HCC formation.

Example 9

This example sets forth several variant peptides having amino acidsubstitutions in the Loop 8 region of FGF19, along with the variants'effect on body weight, certain metabolic parameters, and HCC formation.

The data in Table 10 are associated with variant polypeptides denoted asM3, M139, M140, M141 and M160. The amino acid sequence for M3 is setforth elsewhere herein, and the amino acid sequences for M139, M140,M141 and M160 are as follows:

(SEQ ID NO: 193) RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M139); (SEQ ID NO: 194)RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIREDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M140); (SEQ ID NO: 195)RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILCDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M141); and(SEQ ID NO: 196) RPLAFSDAGPHVHYGWGDPIRQRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (M160).

Only the following sequence domains for each of the aforementionedvariants are listed in Table 10: N-terminal domain, Core, andSheet-8/Loop-8/Sheet-9 region. While the particular amino acid residuesmaking up the Loop 8 region are not universally accepted in theliterature, FGF19 residues 127-129 are defined herein as constitutingthe Loop-8 region.

TABLE 10

    Core Glucose Lower- ing Body Weight Reduction HDL Eleva- tion Tri-glyceride Elevation HCC Forma- tion FGF19 RPLAFSDAGPHVHYGWGDPI RLRHLYTSG//EEIRPDGYNVY// + − + + + (aa 1-20 of SEQ ID (aa 21-29 of (aa 102-112 ofNO: 99) SEQ ID SEQ ID NO: 99) NO: 99) FGF21 HPIPDSSPLLQ--FGGQV RQRYLYTDD//ELLLEDGYNVY// + + − − − (aa 1-20 of SEQ ID (aa 21-29 of (aa 97-107 ofNO: 100) SEQ ID SEQ ID NO: 100) NO: 100) M3 RPLAFSDAGPHVHYGWGDPIRLRHLYTSG //EEILEDGYNVY// + + + + +/− (aa 1-20 of SEQ ID (aa 21-29 of(aa 102-112 of NO: 3) SEQ ID SEQ ID NO: 3) NO: 3) M139RPLAFSDAGPHVHYGWGDPI RLRHLYTSG //EEILEDGYNVY// + − + + +(aa 1-20 of SEQ ID (aa 21-29 of (aa 102-112 of NO: 193) SEQ IDSEQ ID NO: 193) NO: 193) M140 RPLAFSDAGPHVHYGWGDPI RLRHLYTSG//EEILEDGYNVY// + + + + +/− (aa 1-20 of SEQ ID (aa 21-29 of(aa 102-112 of NO: 194) SEQ ID SEQ ID NO: 194) NO: 194) M141RPLAFSDAGPHVHYGWGDPI RLRHLYTSG //EEILEDGYNVY// + − + + +(aa 1-20 of SEQ ID (aa 21-29 of (aa 102-112 of NO: 195) SEQ IDSEQ ID NO: 195) NO: 195) M160 RPLAFSDAGPHVHYGWGDPI RLRHLYTSG//EEILEDGYNVY// + + + + − (aa 1-20 of SEQ ID (aa 21-29 of (aa 102-112 ofNO: 196) SEQ ID SEQ ID  NO: 196) NO: 196)

Referring to Table 10, the P128E substitution appears necessary tosignificantly prevent HCC formation, but is insufficient by itself toprevent HCC formation. In particular, an improvement in preventing HCCformation is observed with the P128E substitution in M140. Conversely,by itself the R127L substitution does not prevent HCC formation (seeM139). As indicated in comparison to M3, a combination of the R127L andP128E substitutions decreases HCC formation but does not eliminate HCCformation. Surprisingly, however, a combination of the R127L and P128Esubstitutions along with a substitution of Gln (Q) for Leu (L) in theFGF19 core region does significantly prevent HCC formation (see M160).

These data indicate that the FGF19 Loop 8 region plays a role in HCCformation. Amino acid residues outside of the Loop 8 region (e.g.,substitutions in the core region) may enhance the prevention of HCCformation.

M1 (SEQ ID NO: 1) RPLAFSDASPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M2 (SEQ ID NO: 2)RPLAFSDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M3 (SEQ ID NO: 3)RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M5 (SEQ ID NO: 5)RHPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M5-R (SEQ ID NO: 160)HPIPDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M48 (SEQ ID NO: 48)RDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M49 (SEQ ID NO: 49)RPLAFSDSSPLLQFGGQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M50 (SEQ ID NO: 50)RHPIPDSSPLLQFGDQVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M51 (SEQ ID NO: 51)RHPIPDSSPLLQFGGNVRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M52 (SEQ ID NO: 52)RDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M53 (SEQ ID NO: 192)MDSSPLLQWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M69 (SEQ ID NO: 69)RDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M70 (SEQ ID NO: 70)MRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M71 (SEQ ID NO: 71)HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHSLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS M72 (SEQ ID NO: 72)HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS M73 (SEQ ID NO: 73)HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVVQDELQGVGGEGCHMHPENCKTLLTDI DRTHTEKPVWDGITGE M75(SEQ ID NO: 75) RVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK M76 (SEQ ID NO: 76)RGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK FGF19 (SEQ ID NO: 99)RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK

Example 10

This example shows that administration of M70 in human patients resultsin suppression of 7a-hydroxy-4-cholsten-3-one (C4), a marker of bileacid synthesis.

Study Subjects:

Healthy adults in the age range 18-65 years and with normal body weight(body mass index, BMI 20-35) were enrolled in the study. The studyprotocol was approved by the Human Research Ethics Committee inAustralia, and written informed consent was obtained from each subject.For inclusion in the study each subject had to be in good healthdetermined by no clinically significant findings from medical history,physical exam, 12 lead ECG, clinical laboratory findings, and vitalsigns at screening. Subjects with history or clinical manifestation ofany significant metabolic, allergic, dermatological, hepatic, renal,hematological, pulmonary, cardiovascular, GI, neurological, orpsychiatric disorder were excluded from enrollment.

Study Design:

The study was a randomized, double-blind, placebo-controlled design.Prescreening of subjects was performed 7-30 days prior to entry, andbaseline evaluations were performed before treatment. Each subject wasgiven subcutaneous injection of M70 at doses 3 mg/day in a single bolusdose daily for 7 days. Blood samples were collected into heparinizedtubes through an indwelling catheter. Blood samples taken on Day 1 andDay 7 at 4.5 hrs or 24 hrs after administration of M70 or placebo wereanalyzed. Serum levels of 7a-hydroxy-4-cholesten-3-one (C4) were used tomonitor CYP7A1 enzymatic activity (bile acid synthesis). They wereanalyzed from individual serum samples after sample extraction followedby high-pressure liquid chromatography (HPLC) as described previously(Galman et al. (2003) J Lipid Res. 2003; 44(4):859-66).

Results:

The data provided in FIG. 6 show that on days 1 and 7, at both 4.5 hoursand 24 hours post-dose, serum levels of C4 were significantly suppressedin the patients, as compared to patients receiving a placebo.

Example 11

This example shows activation of mouse FGFR4-β-klotho signaling byFGF19, M3, and M70 in a rat myoblast cell line

Methods:

An ELK luciferase assay was performed in L6 cells transientlytransfected with mouse FGFR4, b-klotho, and reporter constructscontaining 5×UAS luciferase and GAL4-DNA-binding domain (DBD) fused toELK1. In this system, luciferase activity is regulated by the endogenousphosphorylated extracellular signal-regulated kinase (ERK). Cells wereincubated with ligands for 6 hours before lysed for luciferase activitymeasurements.

A cell-based receptor activation assay was used to evaluate the abilityof mouse FGFR4 to mediate ligand-dependent signaling in the presence ofβ-klotho. To this end, a rat L6 myoblast cell line, which lacksendogenous expression of these proteins, was transfected with DNAsencoding FGFR4 and β-klotho from mouse, as well as plasmids containingan Elk1-dependent chimeric transcription factor-based reporter system.

Following transfection, concentration response of ligand-dependentluciferase expression was analyzed in whole-cell lysates in the presenceof luciferin substrate.

Results:

Co-expression of FGFR4 and β-klotho in L6 cells was found to potentiateactivation of intracellular signaling pathways by both M3, M70 and FGF19(EC₅₀=20, 38 and 53 pM, respectively (see Table 11 and FIG. 7).

TABLE 11 Co-expression of Mouse FGFR4/β-klotho complex in L6 CellsPotentiates Activation of Intracellular Signaling Pathways by FGF19, M3and M70. FGFR4/βklotho Ligand EC₅₀ (pM) E_(max) (fold potentiation)FGF19 52.5 ± 0.01 1.82 ± 0.09 M3 19.8 ± 0.04 1.68 ± 0.04 M70 38.3 ± 0.121.85 ± 0.14 EC₅₀ = half-maximal effective concentration; E_(max) =maximum efficacy. Data are expressed as mean ± SD

These data suggest that the formation of a ternary complex between theFGFR4-β-klotho co-receptors and cognate ligands is important for potentactivation of intracellular signaling.

SEQUENCE LISTING

The present specification is being filed with a computer readable form(CRF) copy of the Sequence Listing. The CRF entitled13370-007_SEQLIST.txt, which was created on Dec. 26, 2013 and is 241,577bytes in size, is identical to the paper copy of the Sequence Listingand is incorporated herein by reference in its entirety.

What is claimed is:
 1. A method of treating a subject havingnonalcoholic steatohepatitis (NASH), comprising administering to thesubject an effective amount of a peptide, wherein said peptidecomprises: a) an N-terminal region comprising at least seven amino acidresidues, the N-terminal region having a first amino acid position and alast amino acid position, wherein the N-terminal region comprises DSSPL(SEQ ID NO:121) or DASPH (SEQ ID NO:122); and b) a C-terminal regionhaving a first amino acid position and a last amino acid position,wherein the C-terminal region comprises (i) a first C-terminal regionsequence comprising WGDPIRLRHLYTSG (amino acids 16 to 29 of SEQ ID NO:99[FGF19]), wherein the W residue corresponds to the first amino acidposition of the C-terminal region; and (ii) a second C-terminal regionsequence comprising PHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (amino acid residues 30 to 194 of SEQ IDNO:99 [FGF19]); or a sequence comprising from 1 to 5 amino acidsubstitutions, deletions or insertions thereof; wherein the peptide (i)binds to fibroblast growth factor receptor 4 (FGFR4) with an affinityequal to or greater than FGF19 binding affinity for FGFR4; (ii)activates FGFR4 to an extent or amount equal to or greater than FGF19activates FGFR4; (iii) has at least one of reduced hepatocellularcarcinoma (HCC) formation; greater glucose lowering activity, less lipidincreasing activity, less triglyceride activity, less cholesterolactivity, less non-HDL activity or less HDL increasing activity, ascompared to FGF19, or as compared to an FGF19 variant sequence havingany of GQV, GDI, WGPI (SEQ ID NO:171), WGDPV (SEQ ID NO:172), WGDI (SEQID NO:173), GDPI (SEQ ID NO:174), GPI, WGQPI (SEQ ID NO:175), WGAPI (SEQID NO:176), AGDPI (SEQ ID NO:177), WADPI (SEQ ID NO:178), WGDAI (SEQ IDNO:179), WGDPA (SEQ ID NO:180), WDPI (SEQ ID NO:181), WGDI (SEQ IDNO:182), WGDP (SEQ ID NO:183) or FGDPI (SEQ ID NO:184), substituted forthe WGDPI (SEQ ID NO:170) sequence at amino acids 16-20 of FGF19 (SEQ IDNO:99); and/or (iv) has less lean mass reducing activity as compared toFGF21; thereby treating said subject.
 2. The method of claim 1, whereinthe second C-terminal region sequence of the peptide comprises from 1 to5 amino acid substitutions, deletions or insertions.
 3. The method ofclaim 1, wherein the peptide is less than about 250 amino acids inlength.
 4. The method of claim 1, wherein the N-terminal regioncomprises amino acid residues DASPHVHYG (SEQ ID NO:102), or DSSPLVHYG(SEQ ID NO:103).
 5. The method of claim 4, wherein the G corresponds tothe last position of the N-terminal region.
 6. The method of claim 5,wherein the N-terminal region further comprises: RHPIP (SEQ ID NO:106),wherein R is the first amino acid position of the N-terminal region;HPIP (SEQ ID NO:107), wherein H is the first amino acid position of theN-terminal region; RPLAF (SEQ ID NO:108), wherein R is the first aminoacid position of the N-terminal region; PLAF (SEQ ID NO:109), wherein Pis the first amino acid position of the N-terminal region; or R, whereinR is the first amino acid position of the N-terminal region.
 7. Themethod of claim 1, wherein the N-terminal region comprises amino acidresidues DSSPLLQ (SEQ ID NO:104), and wherein the Q residue is the lastamino acid position of the N-terminal region.
 8. The method of claim 7,wherein the N-terminal region further comprises: RHPIP (SEQ ID NO:106),wherein R is the first amino acid position of the N-terminal region;HPIP (SEQ ID NO:107), wherein H is the first amino acid position of theN-terminal region; RPLAF (SEQ ID NO:108), wherein R is the first aminoacid position of the N-terminal region; PLAF (SEQ ID NO:109), wherein Pis the first amino acid position of the N-terminal region; or R, whereinR is the first amino acid position of the N-terminal region.
 9. Themethod of claim 1, wherein the N-terminal region comprises amino acidresidues DSSPLLQFGGQV (SEQ ID NO:105), and wherein the V residuecorresponds to the last position of the N-terminal region.
 10. Themethod of claim 1, wherein amino acid residues HPIP (SEQ ID NO:107) arethe first 4 amino acid residues of the N-terminal region.
 11. The methodof claim 1, wherein the first position of the N-terminal region is a Ror M residue; the first and second positions of the N-terminal region isa MR, RM, RD, DS, MD or MS sequence; the first through third positionsof the N-terminal region is a MDS, RDS, MSD, MSS, or DSS sequence; thefirst through fourth positions of the N-terminal region is a RDSS (SEQID NO:115) or MDSS (SEQ ID NO:116) sequence; the first through fifthpositions of the N-terminal region is an MRDSS (SEQ ID NO:117) sequence;the first through sixth positions of the N-terminal region is an MDSSPL(SEQ ID NO:119) sequence; or the first through seventh positions of theN-terminal region is an MSDSSPL (SEQ ID NO:120) sequence.
 12. The methodof claim 1, wherein the peptide comprises an N-terminus region and afirst C-terminal region having an amino acid sequence comprising orconsisting of any of: RPLAFSDASPHVHYGWGDPIRLRHLYTSG (amino acids 1-29 ofSEQ ID NO:1); PLAFSDASPHVHYGWGDPIRLRHLYTSG (amino acids 2-29 of SEQ IDNO:1); RPLAFSDSSPLVHYGWGDPIRLRHLYTSG (amino acids 1-29 of SEQ ID NO:2);PLAFSDSSPLVHYGWGDPIRLRHLYTSG (amino acids 2-29 of SEQ ID NO:2);RHPIPDSSPLLQWGDPIRLRHLYTSG (amino acids 1-26 of SEQ ID NO:8);RHPIPDSSPLLQFGWGDPIRLRHLYTSG (amino acids 1-28 of SEQ ID NO:9);RPLAFSDSSPLVHWGDPIRLRHLYTSG (amino acids 1-27 of SEQ ID NO:26);PLAFSDSSPLVHWGDPIRLRHLYTSG (amino acids 2-27 of SEQ ID NO:26);HPIPDSSPLLQWGDPIRLRHLYTSG (amino acids 1-25 of SEQ ID NO:47);RDSSPLLQWGDPIRLRHLYTSG (amino acids 1-22 of SEQ ID NO:52);DSSPLLQWGDPIRLRHLYTSG (amino acids 2-22 of SEQ ID NO:52);MDSSPLVHYGWGDPIRLRHLYTSG (amino acids 1-24 of SEQ ID NO:53);RDSSPLVHYGWGDPIRLRHLYTSG (amino acids 1-24 of SEQ ID NO:69);DSSPLVHYGWGDPIRLRHLYTSG (amino acids 2-24 of SEQ ID NO:69);MRDSSPLVHYGWGDPIRLRHLYTSG (amino acids 1-25 of SEQ ID NO:70);DSSPLVHYGWGDPIRLRHLYTSG (amino acids 1-23 of SEQ ID NO:141); orHPIPDSSPLLQFGWGDPIRLRHLYTSG (amino acids 1-27 of SEQ ID NO:163).
 13. Themethod of claim 1, wherein the N-terminal region first amino acidposition is a methionine (M), arginine (R), serine (S), histidine (H),proline (P), leucine (L) or aspartic acid (D) residue.
 14. The method ofclaim 1, wherein the N-terminal region does not have a methionine (M) orarginine (R) residue at the first amino acid position of the N-terminalregion.
 15. The method of claim 1, wherein the N-terminal regioncomprises any one of the following amino acid sequences: MDSSPL (SEQ IDNO:119), MSDSSPL (SEQ ID NO:120), or SDSSPL (SEQ ID NO:112).
 16. Themethod of claim 1, wherein the peptide has an amino acid sequencecomprising or consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:8, SEQID NO:9, SEQ ID NO:26, SEQ ID NO:47, SEQ ID NO:52, SEQ ID NO:53, SEQ IDNO:69, SEQ ID NO:70; SEQ ID NO:141 or SEQ ID NO:163.
 17. The method ofclaim 16, wherein the peptide has an amino acid sequence comprising orconsisting of SEQ ID NOs:1, 2, 8, 9, 26, 52 or 69, wherein the arginine(R) residue at the first amino acid position of the N-terminal region ofthe sequence is deleted.
 18. The method of claim 16, wherein the peptidehas an amino acid sequence comprising or consisting of SEQ ID NO:1. 19.The method of claim 16, wherein the peptide has an amino acid sequencecomprising or consisting of SEQ ID NO:2.
 20. The method of claim 16,wherein the peptide has an amino acid sequence comprising or consistingof SEQ ID NO:8.
 21. The method of claim 16, wherein the peptide has anamino acid sequence comprising or consisting of SEQ ID NO:9.
 22. Themethod of claim 16, wherein the peptide has an amino acid sequencecomprising or consisting of SEQ ID NO:26.
 23. The method of claim 16,wherein the peptide has an amino acid sequence comprising or consistingof SEQ ID NO:47.
 24. The method of claim 16, wherein the peptide has anamino acid sequence comprising or consisting of SEQ ID NO:52.
 25. Themethod of claim 16, wherein the peptide has an amino acid sequencecomprising or consisting of SEQ ID NO:53.
 26. The method of claim 16,wherein the peptide has an amino acid sequence comprising or consistingof SEQ ID NO:141.
 27. The method of claim 16, wherein the peptide has anamino acid sequence comprising or consisting of SEQ ID NO:163.
 28. Themethod of claim 1, wherein the second C-terminal region sequencecomprises a EILPD (amino acids 103-107 of SEQ ID NO:193), EIRED (aminoacids 103-107 of SEQ ID NO:194), EILCD (amino acids 103-107 of SEQ IDNO:195), EILED (amino acids 103-107 of SEQ ID NO:196), or LLLED (aminoacids 98-102 of SEQ ID NO:100) sequence substituted for the EIRPDsequence (amino acids 74-78 of SEQ ID NO:188).
 29. The method of claim28, wherein the peptide has an amino acid sequence comprising orconsisting of SEQ ID NO:193.
 30. The method of claim 28, wherein thepeptide has an amino acid sequence comprising or consisting of SEQ IDNO:194.
 31. The method of claim 28, wherein the peptide has an aminoacid sequence comprising or consisting of SEQ ID NO:195.
 32. The methodof claim 1, wherein said peptide is fused with an immunoglobulin Fcregion.
 33. The method of claim 1, wherein the peptide has at least oneof reduced HCC formation; greater glucose lowering activity, or lesslipid increasing activity as compared to FGF19, or as compared to anFGF19 variant having any of GQV, GDI, WGPI (SEQ ID NO:171), WGDPV (SEQID NO:172), WGDI (SEQ ID NO:173), GDPI (SEQ ID NO:174), GPI, WGQPI (SEQID NO:175), WGAPI (SEQ ID NO:176), AGDPI (SEQ ID NO:177), WADPI (SEQ IDNO:178), WGDAI (SEQ ID NO:179), WGDPA (SEQ ID NO:180), WDPI (SEQ IDNO:181), WGDI (SEQ ID NO:182), WGDP (SEQ ID NO:183) or FGDPI (SEQ IDNO:184), substituted for the WGDPI (SEQ ID NO:170) sequence at aminoacids 16-20 of FGF19 (SEQ ID NO:99).
 34. The method of claim 33, whereinthe HCC formation, glucose lowering activity, or lipid increasingactivity is ascertained in a db/db mouse.
 35. The method of claim 33,wherein the peptide has less lean mass reducing activity as compared tothe lean mass reducing activity of FGF21.
 36. The method of claim 33,wherein the lean mass reducing activity is ascertained in a db/db mouse.37. The method of claim 1, wherein the peptide is formulated as apharmaceutical composition further comprising a pharmaceuticallyacceptable carrier.
 38. The method of claim 1 further comprisingadministration of a supplemental therapy.
 39. A method of treating asubject having NASH, comprising administering to the subject aneffective amount of a peptide, wherein said peptide has an amino acidsequence comprising or consisting ofMRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ ID NO:70), or a sequencecomprising a EILPD (amino acids 103-107 of SEQ ID NO:193), EIRED (aminoacids 103-107 of SEQ ID NO:194), EILCD (amino acids 103-107 of SEQ IDNO:195), EILED (amino acids 103-107 of SEQ ID NO:196), or LLLED (aminoacids 98-102 of SEQ ID NO:100) sequence substituted for the EIRPD (aminoacids 99-103 of SEQ ID NO:70) sequence thereof, thereby treating thesubject.
 40. The method of claim 39, wherein the peptide has an aminoacid sequence comprising SEQ ID NO:70.
 41. The method of claim 39,wherein the peptide has an amino acid sequence consisting of SEQ IDNO:70.
 42. The method of claim 39, wherein the amino acid sequencecomprises EILPD (amino acids 103-107 of SEQ ID NO:193), EIRED (aminoacids 103-107 of SEQ ID NO:194), EILCD (amino acids 103-107 of SEQ IDNO:195), EILED (amino acids 103-107 of SEQ ID NO:196), or LLLED (aminoacids 98-102 of SEQ ID NO:100) sequence substituted for the EIRPD (aminoacids 99-103 of SEQ ID NO:70) sequence of SEQ ID NO:70.
 43. The methodof claim 39, wherein said peptide is fused with an immunoglobulin Fcregion.
 44. The method of claim 39, wherein the peptide is formulated asa pharmaceutical composition further comprising a pharmaceuticallyacceptable carrier.
 45. The method of claim 39, further comprisingadministration of a supplemental therapy.
 46. A method of treating asubject having NASH, comprising administering to the subject aneffective amount of a peptide, wherein said peptide has an amino acidsequence comprising or consisting ofRDSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK (SEQ ID NO:69), or a sequencecomprising a EILPD (amino acids 103-107 of SEQ ID NO:193), EIRED (aminoacids 103-107 of SEQ ID NO:194), EILCD (amino acids 103-107 of SEQ IDNO:195), EILED (amino acids 103-107 of SEQ ID NO:196), or LLLED (aminoacids 98-102 of SEQ ID NO:100) sequence substituted for the EIRPD (aminoacids 98-102 of SEQ ID NO:69) sequence thereof, thereby treating thesubject.
 47. The method of claim 46, wherein the peptide has an aminoacid sequence comprising SEQ ID NO:69.
 48. The method of claim 46,wherein the peptide has an amino acid sequence consisting of SEQ IDNO:69.
 49. The method of claim 46, wherein the amino acid sequencecomprises a EILPD (amino acids 103-107 of SEQ ID NO:193), EIRED (aminoacids 103-107 of SEQ ID NO:194), EILCD (amino acids 103-107 of SEQ IDNO:195), EILED (amino acids 103-107 of SEQ ID NO:196), or LLLED (aminoacids 98-102 of SEQ ID NO:100) sequence substituted for the EIRPD (aminoacids 98-102 of SEQ ID NO:69) sequence of SEQ ID NO:69.
 50. The methodof claim 46, wherein said peptide is fused with an immunoglobulin Fcregion.
 51. The method of claim 46, wherein the peptide is formulated asa pharmaceutical composition further comprising a pharmaceuticallyacceptable carrier.
 52. The method of claim 46, further comprisingadministration a supplemental therapy.